Methods and compositions for the treatment of lysosomal storage diseases

ABSTRACT

Nucleases and methods of using these nucleases for inserting a sequence encoding a therapeutic protein such as an enzyme into a cell, thereby providing proteins or cell therapeutics for treatment and/or prevention of a lysosomal storage disease.

CROSS-REFERENCE TO RELATED APPLICATIONS

The present application claims the benefit of U.S. ProvisionalApplication No. 61/670,463, filed Jul. 11, 2012 and U.S. ProvisionalApplication No. 61/704,072, filed Sep. 21, 2012, the disclosures ofwhich are hereby incorporated by reference in their entireties.

TECHNICAL FIELD

The present disclosure is in the field of the treatment of Lysosomalstorage diseases (LSDs) and gene therapy.

BACKGROUND

Gene therapy holds enormous potential for a new era of humantherapeutics. These methodologies will allow treatment for conditionsthat heretofore have not been addressable by standard medical practice.One area that is especially promising is the ability to add a transgeneto a cell to cause that cell to express a product that previously notbeing produced in that cell. Examples of uses of this technology includethe insertion of a gene encoding a therapeutic protein, insertion of acoding sequence encoding a protein that is somehow lacking in the cellor in the individual and insertion of a sequence that encodes astructural nucleic acid such as a microRNA.

Transgenes can be delivered to a cell by a variety of ways, such thatthe transgene becomes integrated into the cell's own genome and ismaintained there. In recent years, a strategy for transgene integrationhas been developed that uses cleavage with site-specific nucleases fortargeted insertion into a chosen genomic locus (see, e.g., co-owned U.S.Pat. No. 7,888,121). Nucleases, such as zinc finger nucleases (ZFNs),transcription activator-like effector nucleases (TALENs), or nucleasesystems such as the CRISPR/Cas system (utilizing an engineered guideRNA), are specific for targeted genes and can be utilized such that thetransgene construct is inserted by either homology directed repair (HDR)or by end capture during non-homologous end joining (NHEJ) drivenprocesses.

Targeted loci include “safe harbor” loci such as the AAVS1, HPRT andCCR5 genes in human cells, and Rosa26 in murine cells (see, e.g.,co-owned United States Patent Publication Nos. 20080299580; 20080159996and 201000218264 and U.S. patent application Ser. No. 13/660,821).Nuclease-mediated integration offers the prospect of improved transgeneexpression, increased safety and expressional durability, as compared toclassic integration approaches that rely on random integration of thetransgene, since it allows exact transgene positioning for a minimalrisk of gene silencing or activation of nearby oncogenes.

While delivery of the transgene to the target cell is one hurdle thatmust be overcome to fully enact this technology, another issue that mustbe conquered is insuring that after the transgene is inserted into thecell and is expressed, the gene product so encoded must reach thenecessary location with the organism, and be made in sufficient localconcentrations to be efficacious. For diseases characterized by the lackof a protein or by the presence of an aberrant non-functional one,delivery of a transgene encoded wild type protein can be extremelyhelpful.

Lysosomal storage diseases (LSDs) are a group of rare metabolicmonogenic diseases characterized by the lack of functional individuallysosomal proteins normally involved in the breakdown of waste lipids,glycoproteins and mucopolysaccharides. These diseases are characterizedby a buildup of these compounds in the cell since it is unable toprocess them for recycling due to the mis-functioning of a specificenzyme. The most common examples are Gaucher's (glucocerebrosidasedeficiency—gene name: GBA), Fabry's (α galactosidase deficiency—GLA),Hunter's (iduronate-2-sulfatase deficiency-IDS), Hurler's (alpha-Liduronidase deficiency—IDUA), and Niemann-Pick's (sphingomyelinphosphodiesterase 1 deficiency—SMPD1) diseases. When grouped alltogether, LSDs have an incidence in the population of about 1 in 7000births. These diseases have devastating effects on those afflicted withthem. They are usually first diagnosed in babies who may havecharacteristic facial and body growth patterns and may have moderate tosevere mental retardation. Treatment options include enzyme replacementtherapy (ERT) where the missing enzyme is given to the patient, usuallythrough intravenous injection in large doses. Such treatment is only totreat the symptoms and is not curative, thus the patient must be givenrepeated dosing of these proteins for the rest of their lives, andpotentially may develop neutralizing antibodies to the injected protein.Often these proteins have a short serum half life, and so the patientmust also endure frequent infusions of the protein. For example,Gaucher's disease patients receiving the Cerezyme® product(imiglucerase) must have infusions three times per week. Production andpurification of the enzymes is also problematic, and so the treatmentsare very costly (>$100,000 per year per patient).

Thus, there remains a need for additional methods and compositions thatcan be used to treat a monogenic disease (e.g. Lysosomal storagediseases) through genome editing, and methods to deliver an expressedtransgene encoded gene product at a therapeutically relevant level.

SUMMARY

Disclosed herein are methods and compositions for treating a monogenicdisease. The invention describes methods for insertion of a transgenesequence into a suitable target cell wherein the transgene encodes aprotein that treats the disease. The therapeutic protein may be excretedfrom the target cell such that it is able to affect or be taken up byother cells that do not harbor the transgene. The invention alsoprovides for methods for the production of a cell (e.g., a mature orundifferentiated cell) that produces high levels of a therapeutic wherethe introduction of a population of these altered cells into a patientwill supply that needed protein to treat a disease or condition.

In one aspect, described herein is a zinc-finger protein (ZFP) thatbinds to target site in a region of interest (e.g., a disease associatedgene, a highly expressed gene, an albumin gene or other or safe harborgene) in a genome, wherein the ZFP comprises one or more engineeredzinc-finger binding domains. In one embodiment, the ZFP is a zinc-fingernuclease (ZFN) that cleaves a target genomic region of interest, whereinthe ZFN comprises one or more engineered zinc-finger binding domains anda nuclease cleavage domain or cleavage half-domain. Cleavage domains andcleavage half domains can be obtained, for example, from variousrestriction endonucleases and/or homing endonucleases. In oneembodiment, the cleavage half-domains are derived from a Type IISrestriction endonuclease (e.g., Fok I). In certain embodiments, the zincfinger domain recognizes a target site in a disease associated or safeharbor gene such as albumin (e.g., a zinc finger protein having 5 or 6fingers with the recognition helix regions shown in a single row ofTable 3).

In another aspect, described herein is a TALE protein (Transcriptionactivator like) that binds to target site in a region of interest (e.g.,a highly expressed gene, a disease associated gene or a safe harborgene) in a genome, wherein the TALE comprises one or more engineeredTALE binding domains. In one embodiment, the TALE is a nuclease (TALEN)that cleaves a target genomic region of interest, wherein the TALENcomprises one or more engineered TALE DNA binding domains and a nucleasecleavage domain or cleavage half-domain. Cleavage domains and cleavagehalf domains can be obtained, for example, from various restrictionendonucleases and/or homing endonucleases. In one embodiment, thecleavage half-domains are derived from a Type IIS restrictionendonuclease (e.g., Fok I). In certain embodiments, the TALE DNA bindingdomain recognizes a target site in a highly expressed, diseaseassociated, or safe harbor gene.

In another aspect, described herein is a CRISPR/Cas system that binds totarget site in a region of interest (e.g., a highly expressed gene, adisease associated gene or a safe harbor gene) in a genome, wherein theCRISPR/Cas system comprises a CRIPSR/Cas nuclease and an engineeredcrRNA/tracrRNA (or single guide RNA). In certain embodiments, theCRISPR/Cas system recognizes a target site in a highly expressed,disease associated, or safe harbor gene.

The ZFN, TALEN, and/or CRISPR/Cas system as described herein may bind toand/or cleave the region of interest in a coding or non-coding regionwithin or adjacent to the gene, such as, for example, a leader sequence,trailer sequence or intron, or within a non-transcribed region, eitherupstream or downstream of the coding region. In certain embodiments, theZFN, TALEN, and/or CRISPR/Cas system binds to and/or cleaves a highlyexpressed gene, for example a globin gene in red blood cells (RBCS).See, e.g., U.S. Application No. 61/670,451, titled “Methods andCompositions for Delivery of Biologics,” filed Jul. 11, 2012,incorporated by reference in its entirety herein. In other embodiments,the ZFN, TALEN, and/or CRISPR/Cas system binds to and/or cleaves asafe-harbor gene, for example a CCR5 gene, a PPP1R12C (also known as AAVS1) gene, albumin, HPRT or a Rosa gene. See, e.g., U.S. PatentPublication Nos. 20080299580; 20080159996 and 201000218264 and U.S.Application Nos. 61/537,349, 61/560,506, 13/660,821 and U.S. ApplicationNo. 61/670,451 titled “Methods and Compositions for Regulation ofTransgene Expression” filed Jul. 11, 2012 and incorporated by referenceherein. In addition, to aid in selection, the HPRT locus may be used(see U.S. patent application Ser. Nos. 13/660,821 and 13/660,843). Inother embodiments, the ZFN, TALEN, and/or CRISPR/Cas system may bind toand/or cleave a disease associated gene (e.g. the gene encodinglysosomal hydrolase α-galactosidase A (AGA), related to Fabry'sDisease). In another aspect, described herein are compositionscomprising one or more of the zinc-finger and/or TALE nucleases orCRISPR/Cas system described herein. Also described are compositionscomprising the one or more of these nucleases and donor nucleic acid. Insome aspects, described are engineered nucleases or CRISPR/Cas systemscapable of cleaving disease associate aberrant regulatory genes andmethods of using these nucleases to treat the disease by reducing oreliminating expression of the aberrant gene product.

In one aspect, the invention describes a method of treating a lysosomalstorage disease by inserting in a corrective transgene into a suitabletarget cell (e.g., blood cell, liver cell, brain cell, stem cell,precursor cell, etc.) such that the product encoded by that correctivetransgene is expressed. In one embodiment, the corrective transgene isinserted into a cell line for the in vitro production of the replacementprotein. The cells comprising the transgene or the protein produced bythe cells can be used to treat a patient in need thereof, for examplefollowing purification of the produced protein. In another embodiment,the corrective transgene is inserted into a target tissue in the bodysuch that the replacement protein is produced in vivo. In some aspects,the expressed protein is excreted from the cell to act on or be taken upby other cells (e.g. via exportation into the blood) that lack thetransgene. In some instances, the target tissue is the liver. In otherinstances, the target tissue is the brain. In other instances, thetarget is blood (e.g., vasculature). In other instances, the target isskeletal muscle. In one embodiment, the corrective gene comprises thewild type sequence of the functioning gene, while in other embodiments,the sequence of the corrective transgene is altered in some manner togive enhanced biological activity. In some aspects, the correctivetransgene comprises optimized codons to increase biological activity,while in other aspects, the sequence is altered to give the resultantprotein more desired function (e.g., improvement in stability,alteration of charge to alter substrate binding etc.). In someembodiments, the transgene is altered for reduced immunogenicity. Inother cases, the transgene is altered such that the encoded proteinbecomes a substrate for transporter-mediated delivery in specifictissues such as the brain (see Gabathuler et al. (2010) Neurobiology ofDisease 37: 48-57).

In another aspect, the invention supplies an engineered nuclease proteincapable of cleaving (editing) the genome of a stem or precursor cell(e.g., blood cell precursor, liver stem cell, etc.) for introduction ofa desired transgene. In some aspects, the edited stem or precursor cellsare then expanded and may be induced to differentiate into a matureedited cells ex vivo, and then the cells are given to the patient. Inother aspects, the edited precursors (e.g., CD34+ stem cells) are givenin a bone marrow transplant which, following successful implantation,proliferate producing edited cells that then differentiate and mature invivo and contain the biologic expressed from the transgene. In otheraspects, the edited stem cells are muscle stem cells which are thenintroduced into muscle tissue. In some aspects, the engineered nucleaseis a Zinc Finger Nuclease (ZFN) and in others, the nuclease is a TALEnuclease (TALEN), and in other aspects, a CRISPR/Cas system is used. Thenucleases may be engineered to have specificity for a safe harbor locus,a gene associated with a disease, or for a gene that is highly expressedin cells. By way of non-limiting example only, the safe harbor locus maybe the AAVS1 site, the CCR5 gene, albumin or the HPRT gene while thedisease associated gene may be the GLA gene encoding lysosomal hydrolaseα-galactosidase A (See Table 2). By way of non-limiting example only, agene that is highly expressed in red blood cells (RBCs) is beta-globin.In another aspect, the transgenic cells are sensitized ex vivo viaelectrosensitization to increase their susceptibility for disruptionfollowing exposure to an energy source (e.g. ultrasound) (see WO2002007752).

In another aspect, described herein is a polynucleotide encoding one ormore ZFN, TALEN, and/or CRISPR/Cas system described herein. Thepolynucleotide may be, for example, mRNA. In some aspects, the mRNA maybe chemically modified (See e.g. Kormann et al, (2011) NatureBiotechnology 29(2):154-157).

In another aspect, described herein is a ZFN, TALEN, and/or CRISPR/Cassystem expression vector comprising a polynucleotide, encoding one ormore ZFN, TALEN, and/or CRISPR/Cas system described herein, operablylinked to a promoter. In one embodiment, the expression vector is aviral vector.

In another aspect, described herein is a host cell comprising one ormore ZFN, TALEN, and/or CRISPR/Cas system expression vectors asdescribed herein. The host cell may be stably transformed or transientlytransfected or a combination thereof with one or more ZFN, TALEN, and/orCRISPR/Cas system expression vectors. In some embodiments, the host cellis a liver cell.

In another aspect, described herein is a method for cleaving a highlyexpressed, disease associated and/or safe harbor locus in a cell, themethod comprising: introducing, into the cell, one or morepolynucleotides encoding one or more ZFN, TALEN, and/or CRISPR/Cassystem that bind(s) to a target site in the one or more target lociunder conditions such that the ZFN(s), TALEN(s) or CRIPSR/Cas system is(are) expressed and the one or more loci are cleaved. Non-limitingexamples of ZFN, TALEN, and/or CRISPR/Cas systems that bind to highlyexpressed and/or safe harbor loci are disclosed in U.S. Publication Nos.20080299580; 20080159996; and 201000218264 and U.S. application Ser.Nos. 13/660,821, 13/660, 843, 13/624,193 and 13/624,217 and U.S.Application No. 61/670,451, titled “Methods and Compositions forDelivery of Biologics,”, all of which are incorporated by reference intheir entireties herein.

In other embodiments, a genomic sequence in any target gene is replacedwith the therapeutic transgene, for example using a ZFN, TALEN, and/orCRISPR/Cas system (or vector encoding said ZFN, TALEN, and/or CRISPR/Cassystem) as described herein and a “donor” sequence or transgene that isinserted into the gene following targeted cleavage with the ZFN, TALEN,and/or CRISPR/Cas system. The donor sequence may be present in the ZFNor TALEN vector, present in a separate vector (e.g., Ad, AAV or LVvector) or, alternatively, may be introduced into the cell using adifferent nucleic acid delivery mechanism. Such insertion of a donornucleotide sequence into the target locus (e.g., highly expressed gene,disease associated gene, other safe-harbor gene, etc.) results in theexpression of the transgene under control of the target locus's (e.g.,albumin, globin, etc.) endogenous genetic control elements. In someaspects, insertion of the transgene of interest, for example into atarget gene (e.g., albumin), results in expression of an intactexogenous protein sequence and lacks any amino acids encoded by thetarget (e.g., albumin). In other aspects, the expressed exogenousprotein is a fusion protein and comprises amino acids encoded by thetransgene and by the endogenous locus into which the transgene isinserted (e.g., from the endogenous target locus or, alternatively fromsequences on the transgene that encode sequences of the target locus).The target may be any gene, for example, a safe harbor gene such as analbumin gene, an AAVS1 gene, an HPRT gene; a CCR5 gene; or a highlyexpressed gene such as a globin gene in an RBC (e.g., beta globin orgamma globin). In some instances, the endogenous sequences will bepresent on the amino (N)-terminal portion of the exogenous protein,while in others, the endogenous sequences will be present on the carboxy(C)-terminal portion of the exogenous protein. In other instances,endogenous sequences will be present on both the N- and C-terminalportions of the exogenous protein. The endogenous sequences may includefull-length wild-type or mutant endogenous sequences or, alternatively,may include partial endogenous amino acid sequences. In someembodiments, the endogenous gene-transgene fusion is located at theendogenous locus within the cell while in other embodiments, theendogenous sequence-transgene coding sequence is inserted into anotherlocus within a genome (e.g., a IDUA-transgene sequence inserted into analbumin, HPRT or CCR5 locus). In some aspects, the safe harbor isselected from the AAVS1, Rosa, albumin, HPRT or CCR5 locus (see co-ownedU.S. Publication Nos. 20080299580; 20080159996; and 201000218264 andU.S. application Ser. Nos. 13/660,821, 13/660, 843, 13/624,193 and13/624,217 and U.S. Application No. 61/670,451, titled “Methods andCompositions for Regulation of Transgene Expression” filed Jul. 11,2011). In other embodiments, the disease associated gene is selectedfrom GLA (lysosomal hydrolase α-galactosidase A), or from one or moregenes listed in Table 2.

In some embodiments the transgene is expressed such that a therapeuticprotein product is retained within the cell (e.g., precursor or maturecell). In other embodiments, the transgene is fused to the extracellulardomain of a membrane protein such that upon expression, a transgenefusion will result in the surface localization of the therapeuticprotein. In some aspects, the extracellular domain is chosen from thoseproteins listed in Table 1. In some aspects, the edited cells alsocomprise a transmembrane protein to traffic the cells to a particulartissue type. In one aspect, the transmemberane protein is a antibody,while in others, the transmembrane protein is a receptor. In certainembodiments, the cell is a precursor (e.g., CD34+ or hematopoietic stemcell) or mature RBC. In some aspects, the therapeutic protein productencoded on the transgene is exported out of the cell to affect or betaken up by cells lacking the transgene. In certain embodiments, thecell is a liver cell which releases the therapeutic protein into theblood stream to act on distal tissues (e.g., brain).

The invention also supplies methods and compositions for the productionof a cell (e.g., RBC) carrying a therapeutic protein for an LSD that canbe used universally for all patients as an allogenic product. This wouldallow the development of a single product for the treatment of patientswith a particular LSD, for example. These carriers may comprisetransmembrane proteins to assist in the trafficking of the cell. In oneaspect, the transmemberane protein is an antibody, while in others, thetransmembrane protein is a receptor.

In one aspect, the invention provides methods and compositions for theknockout of disease associated genes. In some embodiments, these genesare those whose products may regulate expression of a gene in precursoror mature cell. In some aspects, the knock out is to the regulatorytarget site on the DNA for such proteins. In some aspects, the regulatorgene is aberrant such that knock out of the gene restores normalfunction. In other aspects, the gene to be knocked out is a diseaseassociated allele such that the knocking out of this diseased alleleallows expression from a wild type allele and restores normal function.

In one embodiment, the transgene is expressed from the albumin promoterfollowing insertion into the albumin locus. The biologic encoded by thetransgene then may be released into the blood stream if the transgene isinserted into a hepatocyte in vivo. In some aspects, the transgene isdelivered to the liver in vivo in a viral vector through intravenousinjection.

In another embodiment, the transgene encodes a non-coding RNA, e.g. anshRNA. Expression of the transgene prior to cell maturation will resultin a cell containing the non-coding RNA of interest.

In another embodiment, the invention describes precursor cells(hematopoietic stem cells, muscle stem cells or CD34+ hematopoietic stemcell (HSC) cells) into which a transgene has been inserted such thatmature cells derived from these precursors contain high levels of theproduct encoded by the transgene. In some embodiments, these precursorsare induced pluripotent stem cells (iPSC).

In some embodiments, the methods of the invention may be used in vivo intransgenic animal systems. In some aspects, the transgenic animal mayused in model development where the transgene encodes a human gene. Insome instances, the transgenic animal may be knocked out at thecorresponding endogenous locus, allowing the development of an in vivosystem where the human protein may be studied in isolation. Suchtransgenic models may be used for screening purposes to identify smallmolecules, or large biomolecules or other entities which may interactwith or modify the human protein of interest. In some aspects, thetransgene is integrated into the selected locus (e.g., highly expressedor safe-harbor) into a stem cell (e.g., an embryonic stem cell, aninduced pluripotent stem cell, a hepatic stem cell, a neural stem celletc.) or animal embryo obtained by any of the methods described herein,and then the embryo is implanted such that a live animal is born. Theanimal is then raised to sexual maturity and allowed to produceoffspring wherein at least some of the offspring comprise the integratedtransgene.

In a still further aspect, provided herein is a method for site specificintegration of a nucleic acid sequence into an endogenous locus (e.g.,disease-associated, highly expressed such as globin in RBCs, or safeharbor gene such as albumin, CCR5, HPRT or Rosa gene) of a chromosome,for example into the chromosome of an embryo. In certain embodiments,the method comprises: (a) injecting an embryo with (i) at least one DNAvector, wherein the DNA vector comprises an upstream sequence and adownstream sequence flanking the nucleic acid sequence to be integrated,and (ii) at least one RNA molecule encoding a zinc finger, TALE nucleaseor CRISPR/Cas system that recognizes the site of integration in thetarget locus, and (b) culturing the embryo to allow expression of theZFN, TALEN, and/or CRISPR/Cas system, wherein a double stranded breakintroduced into the site of integration by the ZFN, TALEN, and/orCRISPR/Cas system is repaired, via homologous recombination with the DNAvector, so as to integrate the nucleic acid sequence into thechromosome.

In any of the previous embodiments, the methods and compounds of theinvention may be combined with other therapeutic agents for thetreatment of subjects with lysosomal storage diseases. In some aspects,the methods and compositions are used in combination with methods andcompositions to allow passage across the blood brain barrier. In otheraspects, the methods and compositions are used in combination withcompounds known to suppress the immune response of the subject.

A kit, comprising the ZFN, TALEN, and/or CRISPR/Cas system of theinvention, is also provided. The kit may comprise nucleic acids encodingthe ZFN, TALEN, and/or CRISPR/Cas system, (e.g. RNA molecules or theZFN, TALEN, and/or CRISPR/Cas system encoding genes contained in asuitable expression vector), donor molecules, expression vectorsencoding the single guide RNA suitable host cell lines, instructions forperforming the methods of the invention, and the like.

These and other aspects will be readily apparent to the skilled artisanin light of disclosure as a whole.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1, panels A and B, depict a composite set of gels demonstrating theresults of a Cel-I mismatch assay (Surveyor™, Transgenomic) thatmeasures cleavage at a location of interest by a nuclease pair that hasbeen followed by an NHEJ event. NHEJ causes the insertion or deletion ofnucleotide bases (“indels”) which then creates a mismatch when the DNAstrand is annealed with a wild-type DNA strand. FIG. 1A shows theresults measured when the transfection of the albumin-specific nucleasepairs into Neuro2A cells was carried out at 37° C. and FIG. 1B showsresults when transduction of nuclease pairs under hypothermic shock (30°C.). The percent mismatch, or % indels, is a measure of the nucleaseactivity of each pair under each condition.

FIG. 2 is a schematic depicting the structure of four AAV donorsdesigned to provide therapeutic transgenes for treatment of Fabry's,Gaucher's, Hurler's and Hunter's diseases. Each donor construct containsthe AAV sequences (5′ITR and 3′ITR), flanking homology arms forinsertion of the donors into the albumin locus by homology dependentmechanisms, a splice acceptor site, the DNA encoding the replacementenzyme, and a MYC-Flag Tag to allow identification of the integrateddonors.

FIG. 3, panels A and B, demonstrate activity of the mouse albumin ZFNsin vivo. Normal male mice (n=6) were administered a single dose of 200microliter of 1.0×10¹¹ total vector genomes of either AAV2/8 or AAV2/8.2encoding the murine specific ZFN pair SBS30724 and SBS30725 to evaluateliver infectivity by detection of AAV vector genome copies and in vivoNHEJ activity in normal mice. Vectors were given by tail vein injectioninto mice as described, and 14 days post administration, mice weresacrificed, livers harvested and processed for DNA or total proteinquantification. Detection of AAV vector genome copies was performed byquantitative PCR and cleavage activity of the ZFN was measured using theCel-1 (Surveyor, Transkaryotic) assay. FIG. 3A depicts a gel with theCel-I results from mice given AAV2/8 containing a GFP expressioncassette or AAV2/8 comprising the ZFNs, where the AAV were produced viaa 293 expression system or a baculovirus system. FIG. 3B depicts thequantitation of the lanes on the gel and shows that infection of themice with the AAV containing the albumin specific ZFNs results in nearly30% quantitatable NHEJ activity.

FIG. 4, panels A and B, demonstrate the insertion of a huGLa transgenedonor (deficient in patients afflicted with Fabry's disease) into thealbumin locus in mice. FIG. 4A shows a Western blot against the huGLaprotein encoded by the transgene, where the arrow indicates the presumedprotein. Comparison of the mouse samples from those mice that receivedboth ZFN and donor (samples 1-1, 1-2 and 1-3) with the samples thateither received only ZFN (4-1, 4-2, 4-3) or those that only received thehuGLa donor (“hu Fabry donor”), samples 5-1 and 5-2 leads toidentification of a band that coincides with the human liver lysatecontrol. FIG. 4B depicts ELISA results using a huGLa specific ELISA kit,where samples were analyzed from mice either 14 or 30 days followingvirus introduction. Error bars represent standard deviations (n=3). Theresults demonstrate that the mice that received both the ZFN and donor(circles) had higher amounts of huGLa signal that those that onlyreceived ZFN (squares) or only received donor (triangles).

FIG. 5, panels A-D, depicts Western blots that demonstrate expression inliver homogenates of the LSD donor transgenes inserted into the albuminlocus in mice. FIG. 5A shows the results using the IDUA encodingtransgene, FIG. 5B shows the results using the GLA transgene, FIG. 5Cshows the results using the IDS transgene, and FIG. 5D shows the resultsusing the GBA transgene.

FIG. 6 is a schematic displaying the two types of donor insertion thatmay occur following ZFN mediated cleavage. NHEJ mediated donor insertionwill result in the entire LSD-Donor construct being integrated, whereasHDR-mediated insertion will cause only the cDNA including the F9splice-acceptor site to be incorporated. FIG. 6 also depicts thelocation of the two PCR primers (“mALB-OOF1” and “Acc651-SA-rev-sh”)used to detect the type of integration that has occurred.

FIG. 7, panels A-C, depict the results of ³²P radiolabeled PCR performedon liver homogenates on mice containing the integrated LSD transgenes 30days after treatment. FIG. 7A depicts the mice with the IDUA transgene,FIG. 7B depicts those with the GLA transgene, and FIG. 7C depicts thosewith the IDS transgene. In all cases, the bands indicate that insertionof the transgenes has occurred through both NHEJ-mediated andHDR-mediated integration.

FIG. 8 is a schematic illustrating the design of the LSD donorscontaining epitope tags. The location and sequences of the Myc and Flagtags are indicated.

FIG. 9, panels A and B, depicts a gel of ³²P radiolabeled PCR done asdescribed above on liver homogenates from mice with integrated LSDdonors containing the epitope tags. FIG. 9A shows that integrationoccurred through both NHEJ-mediated and HDR-mediated integration for thetargeted GLA, IDUA and IDS transgene.

FIG. 9B shows the same for the GBA transgene.

DETAILED DESCRIPTION

Disclosed herein are methods and compositions for treating or preventinga lysosomal storage disease (LSD). The invention provides methods andcompositions for insertion of a gene encoding a protein that is lackingor insufficiently expressed in the subject with the LSD such that thegene is expressed in the liver and the therapeutic (replacement) proteinis expressed. The invention also describes the alteration of a cell(e.g., precursor or mature RBC, iPSC or liver cell) such that itproduces high levels of the therapeutic and the introduction of apopulation of these altered cells into a patient will supply that neededprotein. The transgene can encode a desired protein or structural RNAthat is beneficial therapeutically in a patient in need thereof.

Thus, the methods and compositions of the invention can be used toexpress from a transgene therapeutically beneficial proteins from anylocus (e.g., highly expressed albumin locus) to replace enzymes that aredefective in lysosomal storage diseases. Additionally, the inventionprovides methods and compositions for treatment of these diseases byinsertion of the sequences into highly expressed loci in cells such asliver cells.

In addition, the transgene can be introduced into patient derived cells,e.g. patient derived induced pluripotent stem cells (iPSCs) or othertypes of stems cells (embryonic or hematopoietic) for use in eventualimplantation. Particularly useful is the insertion of the diseaseassociated transgene into a hematopoietic stem cell for implantationinto a patient in need thereof. As the stem cells differentiate intomature cells, they will contain high levels of the replacement proteinfor delivery to the tissues.

General

Practice of the methods, as well as preparation and use of thecompositions disclosed herein employ, unless otherwise indicated,conventional techniques in molecular biology, biochemistry, chromatinstructure and analysis, computational chemistry, cell culture,recombinant DNA and related fields as are within the skill of the art.These techniques are fully explained in the literature. See, forexample, Sambrook et al. MOLECULAR CLONING: A LABORATORY MANUAL, Secondedition, Cold Spring Harbor Laboratory Press, 1989 and Third edition,2001; Ausubel et al., CURRENT PROTOCOLS IN MOLECULAR BIOLOGY, John Wiley& Sons, New York, 1987 and periodic updates; the series METHODS INENZYMOLOGY, Academic Press, San Diego; Wolffe, CHROMATIN STRUCTURE ANDFUNCTION, Third edition, Academic Press, San Diego, 1998; METHODS INENZYMOLOGY, Vol. 304, “Chromatin” (P. M. Wassarman and A. P. Wolffe,eds.), Academic Press, San Diego, 1999; and METHODS IN MOLECULARBIOLOGY, Vol. 119, “Chromatin Protocols” (P. B. Becker, ed.) HumanaPress, Totowa, 1999.

DEFINITIONS

The terms “nucleic acid,” “polynucleotide,” and “oligonucleotide” areused interchangeably and refer to a deoxyribonucleotide orribonucleotide polymer, in linear or circular conformation, and ineither single- or double-stranded form. For the purposes of the presentdisclosure, these terms are not to be construed as limiting with respectto the length of a polymer. The terms can encompass known analogues ofnatural nucleotides, as well as nucleotides that are modified in thebase, sugar and/or phosphate moieties (e.g., phosphorothioatebackbones). In general, an analogue of a particular nucleotide has thesame base-pairing specificity; i.e., an analogue of A will base-pairwith T.

The terms “polypeptide,” “peptide” and “protein” are usedinterchangeably to refer to a polymer of amino acid residues. The termalso applies to amino acid polymers in which one or more amino acids arechemical analogues or modified derivatives of correspondingnaturally-occurring amino acids.

“Binding” refers to a sequence-specific, non-covalent interactionbetween macromolecules (e.g., between a protein and a nucleic acid). Notall components of a binding interaction need be sequence-specific (e.g.,contacts with phosphate residues in a DNA backbone), as long as theinteraction as a whole is sequence-specific. Such interactions aregenerally characterized by a dissociation constant (K_(d)) of 10⁻⁶ M⁻¹or lower. “Affinity” refers to the strength of binding: increasedbinding affinity being correlated with a lower K_(d).

A “binding protein” is a protein that is able to bind non-covalently toanother molecule. A binding protein can bind to, for example, a DNAmolecule (a DNA-binding protein), an RNA molecule (an RNA-bindingprotein) and/or a protein molecule (a protein-binding protein). In thecase of a protein-binding protein, it can bind to itself (to formhomodimers, homotrimers, etc.) and/or it can bind to one or moremolecules of a different protein or proteins. A binding protein can havemore than one type of binding activity. For example, zinc fingerproteins have DNA-binding, RNA-binding and protein-binding activity.

A “zinc finger DNA binding protein” (or binding domain) is a protein, ora domain within a larger protein, that binds DNA in a sequence-specificmanner through one or more zinc fingers, which are regions of amino acidsequence within the binding domain whose structure is stabilized throughcoordination of a zinc ion. The term zinc finger DNA binding protein isoften abbreviated as zinc finger protein or ZFP.

A “TALE DNA binding domain” or “TALE” is a polypeptide comprising one ormore TALE repeat domains/units. The repeat domains are involved inbinding of the TALE to its cognate target DNA sequence. A single “repeatunit” (also referred to as a “repeat”) is typically 33-35 amino acids inlength and exhibits at least some sequence homology with other TALErepeat sequences within a naturally occurring TALE protein.

Zinc finger and TALE binding domains can be “engineered” to bind to apredetermined nucleotide sequence, for example via engineering (alteringone or more amino acids) of the recognition helix region of a naturallyoccurring zinc finger or TALE protein. Therefore, engineered DNA bindingproteins (zinc fingers or TALEs) are proteins that are non-naturallyoccurring. Non-limiting examples of methods for engineering DNA-bindingproteins are design and selection. A designed DNA binding protein is aprotein not occurring in nature whose design/composition resultsprincipally from rational criteria. Rational criteria for design includeapplication of substitution rules and computerized algorithms forprocessing information in a database storing information of existing ZFPand/or TALE designs and binding data. See, for example, U.S. Pat. Nos.6,140,081; 6,453,242; and 6,534,261; see also WO 98/53058; WO 98/53059;WO 98/53060; WO 02/016536 and WO 03/016496 and U.S. Publication No.20110301073.

A “selected” zinc finger protein or TALE is a protein not found innature whose production results primarily from an empirical process suchas phage display, interaction trap or hybrid selection. See e.g., U.S.Pat. No. 5,789,538; U.S. Pat. No. 5,925,523; U.S. Pat. No. 6,007,988;U.S. Pat. No. 6,013,453; U.S. Pat. No. 6,200,759; WO 95/19431; WO96/06166; WO 98/53057; WO 98/54311; WO 00/27878; WO 01/60970 WO01/88197; WO 02/099084 and U.S. Publication No. 20110301073.

“Recombination” refers to a process of exchange of genetic informationbetween two polynucleotides. For the purposes of this disclosure,“homologous recombination (HR)” refers to the specialized form of suchexchange that takes place, for example, during repair of double-strandbreaks in cells via homology-directed repair mechanisms. This processrequires nucleotide sequence homology, uses a “donor” molecule totemplate repair of a “target” molecule (i.e., the one that experiencedthe double-strand break), and is variously known as “non-crossover geneconversion” or “short tract gene conversion,” because it leads to thetransfer of genetic information from the donor to the target. Withoutwishing to be bound by any particular theory, such transfer can involvemismatch correction of heteroduplex DNA that forms between the brokentarget and the donor, and/or “synthesis-dependent strand annealing,” inwhich the donor is used to re-synthesize genetic information that willbecome part of the target, and/or related processes. Such specialized HRoften results in an alteration of the sequence of the target moleculesuch that part or all of the sequence of the donor polynucleotide isincorporated into the target polynucleotide.

In the methods of the disclosure, one or more targeted nucleases asdescribed herein create a double-stranded break in the target sequence(e.g., cellular chromatin) at a predetermined site, and a “donor”polynucleotide, having homology to the nucleotide sequence in the regionof the break, can be introduced into the cell. The presence of thedouble-stranded break has been shown to facilitate integration of thedonor sequence. The donor sequence may be physically integrated or,alternatively, the donor polynucleotide is used as a template for repairof the break via homologous recombination, resulting in the introductionof all or part of the nucleotide sequence as in the donor into thecellular chromatin. Thus, a first sequence in cellular chromatin can bealtered and, in certain embodiments, can be converted into a sequencepresent in a donor polynucleotide. Thus, the use of the terms “replace”or “replacement” can be understood to represent replacement of onenucleotide sequence by another, (i.e., replacement of a sequence in theinformational sense), and does not necessarily require physical orchemical replacement of one polynucleotide by another.

In any of the methods described herein, additional pairs of zinc-fingeror TALEN proteins can be used for additional double-stranded cleavage ofadditional target sites within the cell.

In certain embodiments of methods for targeted recombination and/orreplacement and/or alteration of a sequence in a region of interest incellular chromatin, a chromosomal sequence is altered by homologousrecombination with an exogenous “donor” nucleotide sequence. Suchhomologous recombination is stimulated by the presence of adouble-stranded break in cellular chromatin, if sequences homologous tothe region of the break are present.

In any of the methods described herein, the first nucleotide sequence(the “donor sequence”) can contain sequences that are homologous, butnot identical, to genomic sequences in the region of interest, therebystimulating homologous recombination to insert a non-identical sequencein the region of interest. Thus, in certain embodiments, portions of thedonor sequence that are homologous to sequences in the region ofinterest exhibit between about 80 to 99% (or any integer therebetween)sequence identity to the genomic sequence that is replaced. In otherembodiments, the homology between the donor and genomic sequence ishigher than 99%, for example if only 1 nucleotide differs as betweendonor and genomic sequences of over 100 contiguous base pairs. Incertain cases, a non-homologous portion of the donor sequence cancontain sequences not present in the region of interest, such that newsequences are introduced into the region of interest. In theseinstances, the non-homologous sequence is generally flanked by sequencesof 50-1,000 base pairs (or any integral value therebetween) or anynumber of base pairs greater than 1,000, that are homologous oridentical to sequences in the region of interest. In other embodiments,the donor sequence is non-homologous to the first sequence, and isinserted into the genome by non-homologous recombination mechanisms.

Any of the methods described herein can be used for partial or completeinactivation of one or more target sequences in a cell by targetedintegration of donor sequence that disrupts expression of the gene(s) ofinterest. Cell lines with partially or completely inactivated genes arealso provided.

Furthermore, the methods of targeted integration as described herein canalso be used to integrate one or more exogenous sequences. The exogenousnucleic acid sequence can comprise, for example, one or more genes orcDNA molecules, or any type of coding or non-coding sequence, as well asone or more control elements (e.g., promoters). In addition, theexogenous nucleic acid sequence may produce one or more RNA molecules(e.g., small hairpin RNAs (shRNAs), inhibitory RNAs (RNAis), microRNAs(miRNAs), etc.).

“Cleavage” refers to the breakage of the covalent backbone of a DNAmolecule. Cleavage can be initiated by a variety of methods including,but not limited to, enzymatic or chemical hydrolysis of a phosphodiesterbond. Both single-stranded cleavage and double-stranded cleavage arepossible, and double-stranded cleavage can occur as a result of twodistinct single-stranded cleavage events. DNA cleavage can result in theproduction of either blunt ends or staggered ends. In certainembodiments, fusion polypeptides are used for targeted double-strandedDNA cleavage.

A “cleavage half-domain” is a polypeptide sequence which, in conjunctionwith a second polypeptide (either identical or different) forms acomplex having cleavage activity (preferably double-strand cleavageactivity). The tennis “first and second cleavage half-domains;” “+ and −cleavage half-domains” and “right and left cleavage half-domains” areused interchangeably to refer to pairs of cleavage half-domains thatdimerize.

An “engineered cleavage half-domain” is a cleavage half-domain that hasbeen modified so as to form obligate heterodimers with another cleavagehalf-domain (e.g., another engineered cleavage half-domain). See, also,U.S. Patent Publication Nos. 2005/0064474, 2007/0218528, 2008/0131962and 2011/0201055, incorporated herein by reference in their entireties.

The term “sequence” refers to a nucleotide sequence of any length, whichcan be DNA or RNA; can be linear, circular or branched and can be eithersingle-stranded or double stranded. The term “donor sequence” refers toa nucleotide sequence that is inserted into a genome. A donor sequencecan be of any length, for example between 2 and 10,000 nucleotides inlength (or any integer value therebetween or thereabove), preferablybetween about 100 and 1,000 nucleotides in length (or any integertherebetween), more preferably between about 200 and 500 nucleotides inlength.

A “disease associated gene” is one that is defective in some manner in amonogenic disease. Non-limiting examples of monogenic diseases includesevere combined immunodeficiency, cystic fibrosis, lysosomal storagediseases (e.g. Gaucher's, Hurler's Hunter's, Fabry's, Neimann-Pick,Tay-Sach's etc), sickle cell anemia, and thalassemia.

“Chromatin” is the nucleoprotein structure comprising the cellulargenome. Cellular chromatin comprises nucleic acid, primarily DNA, andprotein, including histones and non-histone chromosomal proteins. Themajority of eukaryotic cellular chromatin exists in the form ofnucleosomes, wherein a nucleosome core comprises approximately 150 basepairs of DNA associated with an octamer comprising two each of histonesH2A, H2B, H3 and H4; and linker DNA (of variable length depending on theorganism) extends between nucleosome cores. A molecule of histone H1 isgenerally associated with the linker DNA. For the purposes of thepresent disclosure, the term “chromatin” is meant to encompass all typesof cellular nucleoprotein, both prokaryotic and eukaryotic. Cellularchromatin includes both chromosomal and episomal chromatin.

A “chromosome,” is a chromatin complex comprising all or a portion ofthe genome of a cell. The genome of a cell is often characterized by itskaryotype, which is the collection of all the chromosomes that comprisethe genome of the cell. The genome of a cell can comprise one or morechromosomes.

An “episome” is a replicating nucleic acid, nucleoprotein complex orother structure comprising a nucleic acid that is not part of thechromosomal karyotype of a cell. Examples of episomes include plasmidsand certain viral genomes.

A “target site” or “target sequence” is a nucleic acid sequence thatdefines a portion of a nucleic acid to which a binding molecule willbind, provided sufficient conditions for binding exist.

An “exogenous” molecule is a molecule that is not normally present in acell, but can be introduced into a cell by one or more genetic,biochemical or other methods. “Normal presence in the cell” isdetermined with respect to the particular developmental stage andenvironmental conditions of the cell. Thus, for example, a molecule thatis present only during embryonic development of muscle is an exogenousmolecule with respect to an adult muscle cell. Similarly, a moleculeinduced by heat shock is an exogenous molecule with respect to anon-heat-shocked cell. An exogenous molecule can comprise, for example,a functioning version of a malfunctioning endogenous molecule or amalfunctioning version of a normally-functioning endogenous molecule.

An exogenous molecule can be, among other things, a small molecule, suchas is generated by a combinatorial chemistry process, or a macromoleculesuch as a protein, nucleic acid, carbohydrate, lipid, glycoprotein,lipoprotein, polysaccharide, any modified derivative of the abovemolecules, or any complex comprising one or more of the above molecules.Nucleic acids include DNA and RNA, can be single- or double-stranded;can be linear, branched or circular; and can be of any length. Nucleicacids include those capable of forming duplexes, as well astriplex-forming nucleic acids. See, for example, U.S. Pat. Nos.5,176,996 and 5,422,251. Proteins include, but are not limited to,DNA-binding proteins, transcription factors, chromatin remodelingfactors, methylated DNA binding proteins, polymerases, methylases,demethylases, acetylases, deacetylases, kinases, phosphatases,integrases, recombinases, ligases, topoisomerases, gyrases andhelicases.

An exogenous molecule can be the same type of molecule as an endogenousmolecule, e.g., an exogenous protein or nucleic acid. For example, anexogenous nucleic acid can comprise an infecting viral genome, a plasmidor episome introduced into a cell, or a chromosome that is not normallypresent in the cell. Methods for the introduction of exogenous moleculesinto cells are known to those of skill in the art and include, but arenot limited to, lipid-mediated transfer (i.e., liposomes, includingneutral and cationic lipids), electroporation, direct injection, cellfusion, particle bombardment, calcium phosphate co-precipitation,DEAE-dextran-mediated transfer and viral vector-mediated transfer. Anexogenous molecule can also be the same type of molecule as anendogenous molecule but derived from a different species than the cellis derived from. For example, a human nucleic acid sequence may beintroduced into a cell line originally derived from a mouse or hamster.

By contrast, an “endogenous” molecule is one that is normally present ina particular cell at a particular developmental stage under particularenvironmental conditions. For example, an endogenous nucleic acid cancomprise a chromosome, the genome of a mitochondrion, chloroplast orother organelle, or a naturally-occurring episomal nucleic acid.Additional endogenous molecules can include proteins, for example,transcription factors and enzymes.

A “fusion” molecule is a molecule in which two or more subunit moleculesare linked, preferably covalently. The subunit molecules can be the samechemical type of molecule, or can be different chemical types ofmolecules. Examples of the first type of fusion molecule include, butare not limited to, fusion proteins (for example, a fusion between a ZFPor TALE DNA-binding domain and one or more activation domains) andfusion nucleic acids (for example, a nucleic acid encoding the fusionprotein described supra). Examples of the second type of fusion moleculeinclude, but are not limited to, a fusion between a triplex-formingnucleic acid and a polypeptide, and a fusion between a minor groovebinder and a nucleic acid.

Expression of a fusion protein in a cell can result from delivery of thefusion protein to the cell or by delivery of a polynucleotide encodingthe fusion protein to a cell, wherein the polynucleotide is transcribed,and the transcript is translated, to generate the fusion protein.Trans-splicing, polypeptide cleavage and polypeptide ligation can alsobe involved in expression of a protein in a cell. Methods forpolynucleotide and polypeptide delivery to cells are presented elsewherein this disclosure.

A “gene,” for the purposes of the present disclosure, includes a DNAregion encoding a gene product (see infra), as well as all DNA regionswhich regulate the production of the gene product, whether or not suchregulatory sequences are adjacent to coding and/or transcribedsequences. Accordingly, a gene includes, but is not necessarily limitedto, promoter sequences, terminators, translational regulatory sequencessuch as ribosome binding sites and internal ribosome entry sites,enhancers, silencers, insulators, boundary elements, replicationorigins, matrix attachment sites and locus control regions.

“Gene expression” refers to the conversion of the information, containedin a gene, into a gene product. A gene product can be the directtranscriptional product of a gene (e.g., mRNA, tRNA, rRNA, antisenseRNA, ribozyme, structural RNA or any other type of RNA) or a proteinproduced by translation of an mRNA. Gene products also include RNAswhich are modified, by processes such as capping, polyadenylation,methylation, and editing, and proteins modified by, for example,methylation, acetylation, phosphorylation, ubiquitination,ADP-ribosylation, myristilation, and glycosylation.

“Modulation” of gene expression refers to a change in the activity of agene. Modulation of expression can include, but is not limited to, geneactivation and gene repression. Genome editing (e.g., cleavage,alteration, inactivation, random mutation) can be used to modulateexpression. Gene inactivation refers to any reduction in gene expressionas compared to a cell that does not include a ZFP or TALEN as describedherein. Thus, gene inactivation may be partial or complete.

A “region of interest” is any region of cellular chromatin, such as, forexample, a gene or a non-coding sequence within or adjacent to a gene,in which it is desirable to bind an exogenous molecule. Binding can befor the purposes of targeted DNA cleavage and/or targeted recombination.A region of interest can be present in a chromosome, an episome, anorganellar genome (e.g., mitochondrial, chloroplast), or an infectingviral genome, for example. A region of interest can be within the codingregion of a gene, within transcribed non-coding regions such as, forexample, leader sequences, trailer sequences or introns, or withinnon-transcribed regions, either upstream or downstream of the codingregion. A region of interest can be as small as a single nucleotide pairor up to 2,000 nucleotide pairs in length, or any integral value ofnucleotide pairs.

“Eukaryotic” cells include, but are not limited to, fungal cells (suchas yeast), plant cells, animal cells, mammalian cells and human cells(e.g., T-cells).

“Red Blood Cells” (RBCs) or erythrocytes are terminally differentiatedcells derived from hematopoietic stem cells. They lack a nuclease andmost cellular organelles. RBCs contain hemoglobin to carry oxygen fromthe lungs to the peripheral tissues. In fact 33% of an individual RBC ishemoglobin. They also carry CO2 produced by cells during metabolism outof the tissues and back to the lungs for release during exhale. RBCs areproduced in the bone marrow in response to blood hypoxia which ismediated by release of erythropoietin (EPO) by the kidney. EPO causes anincrease in the number of proerythroblasts and shortens the timerequired for full RBC maturation. After approximately 120 days, sincethe RBC do not contain a nucleus or any other regenerative capabilities,the cells are removed from circulation by either the phagocyticactivities of macrophages in the liver, spleen and lymph nodes (˜90%) orby hemolysis in the plasma (˜10%). Following macrophage engulfment,chemical components of the RBC are broken down within vacuoles of themacrophages due to the action of lysosomal enzymes.

“Secretory tissues” are those tissues in an animal that secrete productsout of the individual cell into a lumen of some type which are typicallyderived from epithelium. Examples of secretory tissues that arelocalized to the gastrointestinal tract include the cells that line thegut, the pancreas, and the gallbladder. Other secretory tissues includethe liver, tissues associated with the eye and mucous membranes such assalivary glands, mammary glands, the prostate gland, the pituitary glandand other members of the endocrine system. Additionally, secretorytissues include individual cells of a tissue type which are capable ofsecretion.

The terms “operative linkage” and “operatively linked” (or “operablylinked”) are used interchangeably with reference to a juxtaposition oftwo or more components (such as sequence elements), in which thecomponents are arranged such that both components function normally andallow the possibility that at least one of the components can mediate afunction that is exerted upon at least one of the other components. Byway of illustration, a transcriptional regulatory sequence, such as apromoter, is operatively linked to a coding sequence if thetranscriptional regulatory sequence controls the level of transcriptionof the coding sequence in response to the presence or absence of one ormore transcriptional regulatory factors. A transcriptional regulatorysequence is generally operatively linked in cis with a coding sequence,but need not be directly adjacent to it. For example, an enhancer is atranscriptional regulatory sequence that is operatively linked to acoding sequence, even though they are not contiguous.

With respect to fusion polypeptides, the term “operatively linked” canrefer to the fact that each of the components performs the same functionin linkage to the other component as it would if it were not so linked.For example, with respect to a fusion polypeptide in which a ZFP or TALEDNA-binding domain is fused to an activation domain, the ZFP or TALEDNA-binding domain and the activation domain are in operative linkageif, in the fusion polypeptide, the ZFP or TALE DNA-binding domainportion is able to bind its target site and/or its binding site, whilethe activation domain is able to up-regulate gene expression. When afusion polypeptide in which a ZFP or TALE DNA-binding domain is fused toa cleavage domain, the ZFP or TALE DNA-binding domain and the cleavagedomain are in operative linkage if, in the fusion polypeptide, the ZFPor TALE DNA-binding domain portion is able to bind its target siteand/or its binding site, while the cleavage domain is able to cleave DNAin the vicinity of the target site.

A “functional fragment” of a protein, polypeptide or nucleic acid is aprotein, polypeptide or nucleic acid whose sequence is not identical tothe full-length protein, polypeptide or nucleic acid, yet retains thesame function as the full-length protein, polypeptide or nucleic acid. Afunctional fragment can possess more, fewer, or the same number ofresidues as the corresponding native molecule, and/or can contain one ormore amino acid or nucleotide substitutions. Methods for determining thefunction of a nucleic acid (e.g., coding function, ability to hybridizeto another nucleic acid) are well-known in the art. Similarly, methodsfor determining protein function are well-known. For example, theDNA-binding function of a polypeptide can be determined, for example, byfilter-binding, electrophoretic mobility-shift, or immunoprecipitationassays. DNA cleavage can be assayed by gel electrophoresis. See Ausubelet al., supra. The ability of a protein to interact with another proteincan be determined, for example, by co-immunoprecipitation, two-hybridassays or complementation, both genetic and biochemical. See, forexample, Fields et al. (1989) Nature 340:245-246; U.S. Pat. No.5,585,245 and PCT WO 98/44350.

A “vector” is capable of transferring gene sequences to target cells.Typically, “vector construct,” “expression vector,” and “gene transfervector,” mean any nucleic acid construct capable of directing theexpression of a gene of interest and which can transfer gene sequencesto target cells. Thus, the term includes cloning, and expressionvehicles, as well as integrating vectors.

A “reporter gene” or “reporter sequence” refers to any sequence thatproduces a protein product that is easily measured, preferably althoughnot necessarily in a routine assay. Suitable reporter genes include, butare not limited to, sequences encoding proteins that mediate antibioticresistance (e.g., ampicillin resistance, neomycin resistance, G418resistance, puromycin resistance), sequences encoding colored orfluorescent or luminescent proteins (e.g., green fluorescent protein,enhanced green fluorescent protein, red fluorescent protein,luciferase), and proteins which mediate enhanced cell growth and/or geneamplification (e.g., dihydrofolate reductase). Epitope tags include, forexample, one or more copies of FLAG, His, myc, Tap, HA or any detectableamino acid sequence. “Expression tags” include sequences that encodereporters that may be operably linked to a desired gene sequence inorder to monitor expression of the gene of interest.

The terms “subject” and “patient” are used interchangeably and refer tomammals such as human patients and non-human primates, as well asexperimental animals such as rabbits, dogs, cats, rats, mice, and otheranimals. Accordingly, the term “subject” or “patient” as used hereinmeans any mammalian patient or subject to which the altered cells of theinvention and/or proteins produced by the altered cells of the inventioncan be administered. Subjects of the present invention include thosehaving an LSD.

Nucleases

Described herein are compositions, particularly nucleases, which areuseful targeting a gene for the insertion of a transgene, for example,nucleases that are specific for a safe-harbor gene such as albumin. Incertain embodiments, the nuclease is naturally occurring. In otherembodiments, the nuclease is non-naturally occurring, i.e., engineeredin the DNA-binding domain and/or cleavage domain. For example, theDNA-binding domain of a naturally-occurring nuclease or nuclease systemmay be altered to bind to a selected target site (e.g., a meganucleasethat has been engineered to bind to site different than the cognatebinding site or a CRISPR/Cas system utilizing an engineered single guideRNA). In other embodiments, the nuclease comprises heterologousDNA-binding and cleavage domains (e.g., zinc finger nucleases;TAL-effector nucleases; meganuclease DNA-binding domains withheterologous cleavage domains).

A. DNA-Binding Domains

In certain embodiments, the nuclease is a meganuclease (homingendonuclease). Naturally-occurring meganucleases recognize 15-40base-pair cleavage sites and are commonly grouped into four families:the LAGLIDADG family, the GIY-YIG family, the His-Cyst box family andthe HNH family. Exemplary homing endonucleases include I-SceI, I-CeuI,PI-PspI, PI-Sce, I-SceIV, I-CsmI, I-PanI, I-SceII, I-PpoI, I-SceIII,I-CreI, I-TevI, I-TevII and I-TevIII. Their recognition sequences areknown. See also U.S. Pat. No. 5,420,032; U.S. Pat. No. 6,833,252;Belfort et al. (1997) Nucleic Acids Res. 25:3379-3388; Dujon et al.(1989) Gene 82:115-118; Perler et al. (1994) Nucleic Acids Res. 22,1125-1127; Jasin (1996) Trends Genet. 12:224-228; Gimble et al. (1996)J. Mol. Biol. 263:163-180; Argast et al. (1998) J. Mol. Biol.280:345-353 and the New England Biolabs catalogue.

In certain embodiments, the nuclease comprises an engineered(non-naturally occurring) homing endonuclease (meganuclease). Therecognition sequences of homing endonucleases and meganucleases such asI-SceI, I-CeuI, PI-PspI, PI-Sce, I-SceIV , I-CsmI, I-SceIII, I-CreI,I-TevI, I-TevII and I-TevIII are known. See also U.S. Pat. No.5,420,032; U.S. Pat. No. 6,833,252; Belfort et al. (1997) Nucleic AcidsRes. 25:3379-3388; Dujon et al. (1989) Gene 82:115-118; Perler et al.(1994) Nucleic Acids Res. 22, 1125-1127; Jasin (1996) Trends Genet.12:224-228; Gimble et al. (1996) J. Mol. Biol. 263:163-180; Argast etal. (1998) J. Mol. Biol. 280:345-353 and the New England Biolabscatalogue. In addition, the DNA-binding specificity of homingendonucleases and meganucleases can be engineered to bind non-naturaltarget sites. See, for example, Chevalier et al. (2002) Molec. Cell10:895-905; Epinat et al. (2003) Nucleic Acids Res. 31:2952-2962;Ashworth et al. (2006) Nature 441:656-659; Paques et al. (2007) CurrentGene Therapy 7:49-66; U.S. Patent Publication No. 20070117128. TheDNA-binding domains of the homing endonucleases and meganucleases may bealtered in the context of the nuclease as a whole (i.e., such that thenuclease includes the cognate cleavage domain) or may be fused to aheterologous cleavage domain.

In other embodiments, the DNA-binding domain comprises a naturallyoccurring or engineered (non-naturally occurring) TAL effector DNAbinding domain. See, e.g., U.S. Patent Publication No. 20110301073,incorporated by reference in its entirety herein. The plant pathogenicbacteria of the genus Xanthomonas are known to cause many diseases inimportant crop plants. Pathogenicity of Xanthomonas depends on aconserved type III secretion (T3S) system which injects more than 25different effector proteins into the plant cell. Among these injectedproteins are transcription activator-like effectors (TALE) which mimicplant transcriptional activators and manipulate the plant transcriptome(see Kay et al (2007) Science 318:648-651). These proteins contain a DNAbinding domain and a transcriptional activation domain. One of the mostwell characterized TALEs is AvrBs3 from Xanthomonas campestgris pv.Vesicatoria (see Bonas et al (1989) Mol Gen Genet 218: 127-136 andWO2010079430). TALEs contain a centralized domain of tandem repeats,each repeat containing approximately 34 amino acids, which are key tothe DNA binding specificity of these proteins. In addition, they containa nuclear localization sequence and an acidic transcriptional activationdomain (for a review see Schornack S, et at (2006) J Plant Physiol163(3): 256-272). In addition, in the phytopathogenic bacteria Ralstoniasolanacearum two genes, designated brg11 and hpx17 have been found thatare homologous to the AvrBs3 family of Xanthomonas in the R.solanacearum biovar 1 strain GMI1000 and in the biovar 4 strain RS1000(See Heuer et al (2007) Appl and Envir Micro 73(13): 4379-4384). Thesegenes are 98.9% identical in nucleotide sequence to each other butdiffer by a deletion of 1,575 bp in the repeat domain of hpx17. However,both gene products have less than 40% sequence identity with AvrBs3family proteins of Xanthomonas.

Thus, in some embodiments, the DNA binding domain that binds to a targetsite in a target locus (e.g., albumin or other safe harbor) is anengineered domain from a TAL effector similar to those derived from theplant pathogens Xanthomonas (see Boch et al, (2009) Science 326:1509-1512 and Moscou and Bogdanove, (2009) Science 326: 1501) andRalstonia (see Heuer et al (2007) Applied and Environmental Microbiology73(13): 4379-4384); U.S. Patent Publication Nos. 20110301073 and20110145940. See, e.g., albumin TALENs in U.S. application Ser. No.13/624,193 and 13/624,217, incorporated by reference herein in theirentireties.

In certain embodiments, the DNA binding domain comprises a zinc fingerprotein (e.g., a zinc finger protein that binds to a target site in analbumin or safe-harbor gene). Preferably, the zinc finger protein isnon-naturally occurring in that it is engineered to bind to a targetsite of choice. See, for example, See, for example, Beerli et al. (2002)Nature Biotechnol. 20:135-141; Pabo et al. (2001) Ann. Rev. Biochem.70:313-340; Isalan et al. (2001) Nature Biotechnol. 19:656-660; Segal etal. (2001) Curr. Opin. Biotechnol. 12:632-637; Choo et al. (2000) Curr.Opin. Struct. Biol. 10:411-416; U.S. Pat. Nos. 6,453,242; 6,534,261;6,599,692; 6,503,717; 6,689,558; 7,030,215; 6,794,136; 7,067,317;7,262,054; 7,070,934; 7,361,635; 7,253,273; and U.S. Patent PublicationNos. 2005/0064474; 2007/0218528; 2005/0267061, all incorporated hereinby reference in their entireties.

An engineered zinc finger binding or TALE domain can have a novelbinding specificity, compared to a naturally-occurring zinc fingerprotein. Engineering methods include, but are not limited to, rationaldesign and various types of selection. Rational design includes, forexample, using databases comprising triplet (or quadruplet) nucleotidesequences and individual zinc finger amino acid sequences, in which eachtriplet or quadruplet nucleotide sequence is associated with one or moreamino acid sequences of zinc fingers which bind the particular tripletor quadruplet sequence. See, for example, co-owned U.S. Pat. Nos.6,453,242 and 6,534,261, incorporated by reference herein in theirentireties.

Exemplary selection methods, including phage display and two-hybridsystems, are disclosed in U.S. Pat. Nos. 5,789,538; 5,925,523;6,007,988; 6,013,453; 6,410,248; 6,140,466; 6,200,759; and 6,242,568; aswell as WO 98/37186; WO 98/53057; WO 00/27878; WO 01/88197 and GB2,338,237. In addition, enhancement of binding specificity for zincfinger binding domains has been described, for example, in co-owned WO02/077227.

In addition, as disclosed in these and other references, DNA domains(e.g., multi-fingered zinc finger proteins or TALE domains) may belinked together using any suitable linker sequences, including forexample, linkers of 5 or more amino acids in length. See, also, U.S.Pat. Nos. 6,479,626; 6,903,185; and 7,153,949 for exemplary linkersequences 6 or more amino acids in length. The DNA binding proteinsdescribed herein may include any combination of suitable linkers betweenthe individual zinc fingers of the protein. In addition, enhancement ofbinding specificity for zinc finger binding domains has been described,for example, in co-owned WO 02/077227.

Selection of target sites; DNA-binding domains and methods for designand construction of fusion proteins (and polynucleotides encoding same)are known to those of skill in the art and described in detail in U.S.Pat. Nos. 6,140,0815; 789,538; 6,453,242; 6,534,261; 5,925,523;6,007,988; 6,013,453; 6,200,759; WO 95/19431; WO 96/06166; WO 98/53057;WO 98/54311; WO 00/27878; WO 01/60970 WO 01/88197; WO 02/099084; WO98/53058; WO 98/53059; WO 98/53060; WO 02/016536 and WO 03/016496 andU.S. Publication No. 20110301073.

In addition, as disclosed in these and other references, DNA-bindingdomains (e.g., multi-fingered zinc finger proteins) may be linkedtogether using any suitable linker sequences, including for example,linkers of 5 or more amino acids in length. See, also, U.S. Pat. Nos.6,479,626; 6,903,185; and 7,153,949 for exemplary linker sequences 6 ormore amino acids in length. The proteins described herein may includeany combination of suitable linkers between the individual zinc fingersof the protein.

B. Cleavage Domains

Any suitable cleavage domain can be operatively linked to a DNA-bindingdomain to form a nuclease. For example, ZFP DNA-binding domains havebeen fused to nuclease domains to create ZFNs—a functional entity thatis able to recognize its intended nucleic acid target through itsengineered (ZFP) DNA binding domain and cause the DNA to be cut near theZFP binding site via the nuclease activity. See, e.g., Kim et al. (1996)Proc Nat'l Acad Sci USA 93(3):1156-1160. More recently, ZFNs have beenused for genome modification in a variety of organisms. See, forexample, United States Patent Publications 20030232410; 20050208489;20050026157; 20050064474; 20060188987; 20060063231; and InternationalPublication WO 07/014,275. Likewise, TALE DNA-binding domains have beenfused to nuclease domains to create TALENs. See, e.g., U.S. PublicationNo. 20110301073.

As noted above, the cleavage domain may be heterologous to theDNA-binding domain, for example a zinc finger DNA-binding domain and acleavage domain from a nuclease or a TALEN DNA-binding domain and acleavage domain, or meganuclease DNA-binding domain and cleavage domainfrom a different nuclease. Heterologous cleavage domains can be obtainedfrom any endonuclease or exonuclease. Exemplary endonucleases from whicha cleavage domain can be derived include, but are not limited to,restriction endonucleases and homing endonucleases. See, for example,2002-2003 Catalogue, New England Biolabs, Beverly, Mass.; and Belfort etal. (1997) Nucleic Acids Res. 25:3379-3388. Additional enzymes whichcleave DNA are known (e.g., S1 Nuclease; mung bean nuclease; pancreaticDNase I; micrococcal nuclease; yeast HO endonuclease; see also Linn etal. (eds.) Nucleases, Cold Spring Harbor Laboratory Press, 1993). One ormore of these enzymes (or functional fragments thereof) can be used as asource of cleavage domains and cleavage half-domains.

Similarly, a cleavage half-domain can be derived from any nuclease orportion thereof, as set forth above, that requires dimerization forcleavage activity. In general, two fusion proteins are required forcleavage if the fusion proteins comprise cleavage half-domains.Alternatively, a single protein comprising two cleavage half-domains canbe used. The two cleavage half-domains can be derived from the sameendonuclease (or functional fragments thereof), or each cleavagehalf-domain can be derived from a different endonuclease (or functionalfragments thereof). In addition, the target sites for the two fusionproteins are preferably disposed, with respect to each other, such thatbinding of the two fusion proteins to their respective target sitesplaces the cleavage half-domains in a spatial orientation to each otherthat allows the cleavage half-domains to form a functional cleavagedomain, e.g., by dimerizing. Thus, in certain embodiments, the nearedges of the target sites are separated by 5-8 nucleotides or by 15-18nucleotides. However any integral number of nucleotides or nucleotidepairs can intervene between two target sites (e.g., from 2 to 50nucleotide pairs or more). In general, the site of cleavage lies betweenthe target sites.

Restriction endonucleases (restriction enzymes) are present in manyspecies and are capable of sequence-specific binding to DNA (at arecognition site), and cleaving DNA at or near the site of binding.Certain restriction enzymes (e.g., Type IIS) cleave DNA at sites removedfrom the recognition site and have separable binding and cleavagedomains For example, the Type IIS enzyme Fok I catalyzes double-strandedcleavage of DNA, at 9 nucleotides from its recognition site on onestrand and 13 nucleotides from its recognition site on the other. See,for example, U.S. Pat. Nos. 5,356,802; 5,436,150 and 5,487,994; as wellas Li et al. (1992) Proc. Natl. Acad. Sci. USA 89:4275-4279; Li et al.(1993) Proc. Natl. Acad. Sci. USA 90:2764-2768; Kim et al. (1994a) Proc.Natl. Acad. Sci. USA 91:883-887; Kim et al. (1994b) J. Biol. Chem.269:31,978-31,982. Thus, in one embodiment, fusion proteins comprise thecleavage domain (or cleavage half-domain) from at least one Type IISrestriction enzyme and one or more zinc finger binding domains, whichmay or may not be engineered.

An exemplary Type IIS restriction enzyme, whose cleavage domain isseparable from the binding domain, is Fok I. This particular enzyme isactive as a dimer Bitinaite et al. (1998) Proc. Natl. Acad. Sci. USA 95:10,570-10,575. Accordingly, for the purposes of the present disclosure,the portion of the Fok I enzyme used in the disclosed fusion proteins isconsidered a cleavage half-domain. Thus, for targeted double-strandedcleavage and/or targeted replacement of cellular sequences using zincfinger-Fok I fusions, two fusion proteins, each comprising a FokIcleavage half-domain, can be used to reconstitute a catalytically activecleavage domain. Alternatively, a single polypeptide molecule containinga DNA binding domain and two Fok I cleavage half-domains can also beused.

A cleavage domain or cleavage half-domain can be any portion of aprotein that retains cleavage activity, or that retains the ability tomultimerize (e.g., dimerize) to form a functional cleavage domain.

Exemplary Type IIS restriction enzymes are described in InternationalPublication WO 07/014,275, incorporated herein in its entirety.Additional restriction enzymes also contain separable binding andcleavage domains, and these are contemplated by the present disclosure.See, for example, Roberts et al. (2003) Nucleic Acids Res. 31:418-420.

In certain embodiments, the cleavage domain comprises one or moreengineered cleavage half-domain (also referred to as dimerization domainmutants) that minimize or prevent homodimerization, as described, forexample, in U.S. Patent Publication Nos. 20050064474; 20060188987 and20080131962, the disclosures of all of which are incorporated byreference in their entireties herein Amino acid residues at positions446, 447, 479, 483, 484, 486, 487, 490, 491, 496, 498, 499, 500, 531,534, 537, and 538 of Fok I are all targets for influencing dimerizationof the Fok I cleavage half-domains.

Exemplary engineered cleavage half-domains of Fok I that faun obligateheterodimers include a pair in which a first cleavage half-domainincludes mutations at amino acid residues at positions 490 and 538 ofFok I and a second cleavage half-domain includes mutations at amino acidresidues 486 and 499.

Thus, in one embodiment, a mutation at 490 replaces Glu (E) with Lys(K); the mutation at 538 replaces Iso (I) with Lys (K); the mutation at486 replaced Gln (Q) with Glu (E); and the mutation at position 499replaces Iso (I) with Lys (K). Specifically, the engineered cleavagehalf-domains described herein were prepared by mutating positions 490(E→K) and 538 (I→K) in one cleavage half-domain to produce an engineeredcleavage half-domain designated “E490K:I538K” and by mutating positions486 (Q→E) and 499 (I→L) in another cleavage half-domain to produce anengineered cleavage half-domain designated “Q486E:I499L”. The engineeredcleavage half-domains described herein are obligate heterodimer mutantsin which aberrant cleavage is minimized or abolished. See, e.g., U.S.Patent Publication No. 2008/0131962, the disclosure of which isincorporated by reference in its entirety for all purposes.

In certain embodiments, the engineered cleavage half-domain comprisesmutations at positions 486, 499 and 496 (numbered relative to wild-typeFold), for instance mutations that replace the wild type Gln (Q) residueat position 486 with a Glu (E) residue, the wild type Iso (I) residue atposition 499 with a Leu (L) residue and the wild-type Asn (N) residue atposition 496 with an Asp (D) or Glu (E) residue (also referred to as a“ELD” and “ELE” domains, respectively). In other embodiments, theengineered cleavage half-domain comprises mutations at positions 490,538 and 537 (numbered relative to wild-type FokI), for instancemutations that replace the wild type Glu (E) residue at position 490with a Lys (K) residue, the wild type Iso (I) residue at position 538with a Lys (K) residue, and the wild-type His (H) residue at position537 with a Lys (K) residue or a Arg (R) residue (also referred to as“KKK” and “KKR” domains, respectively). In other embodiments, theengineered cleavage half-domain comprises mutations at positions 490 and537 (numbered relative to wild-type Fold), for instance mutations thatreplace the wild type Glu (E) residue at position 490 with a Lys (K)residue and the wild-type His (H) residue at position 537 with a Lys (K)residue or a Arg (R) residue (also referred to as “KIK” and “KIR”domains, respectively). (See US Patent Publication No. 20110201055).Engineered cleavage half-domains described herein can be prepared usingany suitable method, for example, by site-directed mutagenesis ofwild-type cleavage half-domains (Fok I) as described in U.S. PatentPublication Nos. 20050064474; 20080131962; and 20110201055.

Alternatively, nucleases may be assembled in vivo at the nucleic acidtarget site using so-called “split-enzyme” technology (see e.g. U.S.Patent Publication No. 20090068164). Components of such split enzymesmay be expressed either on separate expression constructs, or can belinked in one open reading frame where the individual components areseparated, for example, by a self-cleaving 2A peptide or IRES sequence.Components may be individual zinc finger binding domains or domains of ameganuclease nucleic acid binding domain.

Nucleases can be screened for activity prior to use, for example in ayeast-based chromosomal system as described in WO 2009/042163 and20090068164. Nuclease expression constructs can be readily designedusing methods known in the art. See, e.g., United States PatentPublications 20030232410; 20050208489; 20050026157; 20050064474;20060188987; 20060063231; and International Publication WO 07/014,275.Expression of the nuclease may be under the control of a constitutivepromoter or an inducible promoter, for example the galactokinasepromoter which is activated (de-repressed) in the presence of raffinoseand/or galactose and repressed in presence of glucose.

The CRISPR (Clustered Regularly Interspaced Short PalindromicRepeats)/Cas (CRISPR Associated) nuclease system is a recentlyengineered nuclease system based on a bacterial system that can be usedfor genome engineering. It is based on part of the adaptive immuneresponse of many bacteria and archea. When a virus or plasmid invades abacterium, segments of the invader's DNA are converted into CRISPR RNAs(crRNA) by the ‘immune’ response. This crRNA then associates, through aregion of partial complementarity, with another type of RNA calledtracrRNA to guide the Cas9 nuclease to a region homologous to the crRNAin the target DNA called a “protospacer”. Cas9 cleaves the DNA togenerate blunt ends at the DSB at sites specified by a 20-nucleotideguide sequence contained within the crRNA transcript. Cas9 requires boththe crRNA and the tracrRNA for site specific DNA recognition andcleavage. This system has now been engineered such that the crRNA andtracrRNA can be combined into one molecule (the “single guide RNA”); andthe crRNA equivalent portion of the single guide RNA can be engineeredto guide the Cas9 nuclease to target any desired sequence (see Jinek etal (2012) Science 337, p. 816-821, Jinek et al, (2013), eLife 2:e00471,and David Segal, (2013) eLife 2:e00563). Thus, the CRISPR/Cas system canbe engineered to create a DSB at a desired target in a genome, andrepair of the DSB can be influenced by the use of repair inhibitors tocause an increase in error prone repair.

Target Sites

As described in detail above, DNA domains can be engineered to bind toany sequence of choice in a locus, for example an albumin or othersafe-harbor gene. An engineered DNA-binding domain can have a novelbinding specificity, compared to a naturally-occurring DNA-bindingdomain Engineering methods include, but are not limited to, rationaldesign and various types of selection. Rational design includes, forexample, using databases comprising triplet (or quadruplet) nucleotidesequences and individual (e.g., zinc finger) amino acid sequences, inwhich each triplet or quadruplet nucleotide sequence is associated withone or more amino acid sequences of DNA binding domain which bind theparticular triplet or quadruplet sequence. See, for example, co-ownedU.S. Pat. Nos. 6,453,242 and 6,534,261, incorporated by reference hereinin their entireties. Rational design of TAL-effector domains can also beperformed. See, e.g., U.S. Publication No. 20110301073.

Exemplary selection methods applicable to DNA-binding domains, includingphage display and two-hybrid systems, are disclosed in U.S. Pat. Nos.5,789,538; 5,925,523; 6,007,988; 6,013,453; 6,410,248; 6,140,466;6,200,759; and 6,242,568; as well as WO 98/37186; WO 98/53057; WO00/27878; WO 01/88197 and GB 2,338,237.

Selection of target sites; nucleases and methods for design andconstruction of fusion proteins (and polynucleotides encoding same) areknown to those of skill in the art and described in detail in U.S.Patent Application Publication Nos. 20050064474 and 20060188987,incorporated by reference in their entireties herein.

In addition, as disclosed in these and other references, DNA-bindingdomains (e.g., multi-fingered zinc finger proteins) may be linkedtogether using any suitable linker sequences, including for example,linkers of 5 or more amino acids. See, e.g., U.S. Pat. Nos. 6,479,626;6,903,185; and 7,153,949 for exemplary linker sequences 6 or more aminoacids in length. The proteins described herein may include anycombination of suitable linkers between the individual DNA-bindingdomains of the protein. See, also, U.S. Publication No. 20110301073.

Donors

As noted above, insertion of an exogenous sequence (also called a “donorsequence” or “donor”), for example for correction of a mutant gene orfor increased expression of a wild-type gene. It will be readilyapparent that the donor sequence is typically not identical to thegenomic sequence where it is placed. A donor sequence can contain anon-homologous sequence flanked by two regions of homology to allow forefficient HDR at the location of interest. Additionally, donor sequencescan comprise a vector molecule containing sequences that are nothomologous to the region of interest in cellular chromatin. A donormolecule can contain several, discontinuous regions of homology tocellular chromatin. For example, for targeted insertion of sequences notnormally present in a region of interest, said sequences can be presentin a donor nucleic acid molecule and flanked by regions of homology tosequence in the region of interest.

The donor polynucleotide can be DNA or RNA, single-stranded ordouble-stranded and can be introduced into a cell in linear or circularform. If introduced in linear form, the ends of the donor sequence canbe protected (e.g., from exonucleolytic degradation) by methods known tothose of skill in the art. For example, one or more dideoxynucleotideresidues are added to the 3′ terminus of a linear molecule and/orself-complementary oligonucleotides are ligated to one or both ends.See, for example, Chang et al. (1987) Proc. Natl. Acad. Sci. USA84:4959-4963; Nehis et al. (1996) Science 272:886-889. Additionalmethods for protecting exogenous polynucleotides from degradationinclude, but are not limited to, addition of terminal amino group(s) andthe use of modified internucleotide linkages such as, for example,phosphorothioates, phosphoramidates, and O-methyl ribose or deoxyriboseresidues.

A polynucleotide can be introduced into a cell as part of a vectormolecule having additional sequences such as, for example, replicationorigins, promoters and genes encoding antibiotic resistance. Moreover,donor polynucleotides can be introduced as naked nucleic acid, asnucleic acid complexed with an agent such as a liposome or poloxamer, orcan be delivered by viruses (e.g., adenovirus, AAV, herpesvirus,retrovirus, lentivirus and integrase defective lentivirus (IDLV)).

The donor is generally inserted so that its expression is driven by theendogenous promoter at the integration site, namely the promoter thatdrives expression of the endogenous gene into which the donor isinserted (e.g., highly expressed, albumin, AAVS1, HPRT, etc.). However,it will be apparent that the donor may comprise a promoter and/orenhancer, for example a constitutive promoter or an inducible or tissuespecific promoter.

The donor molecule may be inserted into an endogenous gene such thatall, some or none of the endogenous gene is expressed. For example, atransgene as described herein may be inserted into an albumin or otherlocus such that some (N-terminal and/or C-terminal to the transgeneencoding the lysosomal enzyme) or none of the endogenous albuminsequences are expressed, for example as a fusion with the transgeneencoding the lysosomal sequences. In other embodiments, the transgene(e.g., with or without additional coding sequences such as for albumin)is integrated into any endogenous locus, for example a safe-harborlocus. See, e.g., U.S. patent publications 20080299580; 20080159996 and201000218264.

When endogenous sequences (endogenous or part of the transgene) areexpressed with the transgene, the endogenous sequences (e.g., albumin,etc.) may be full-length sequences (wild-type or mutant) or partialsequences. Preferably the endogenous sequences are functional.Non-limiting examples of the function of these full length or partialsequences (e.g., albumin) include increasing the serum half-life of thepolypeptide expressed by the transgene (e.g., therapeutic gene) and/oracting as a carrier.

Furthermore, although not required for expression, exogenous sequencesmay also include transcriptional or translational regulatory sequences,for example, promoters, enhancers, insulators, internal ribosome entrysites, sequences encoding 2A peptides and/or polyadenylation signals.

In certain embodiments, the exogenous sequence (donor) comprises afusion of a protein of interest and, as its fusion partner, anextracellular domain of a membrane protein, causing the fusion proteinto be located on the surface of the cell. This allows the proteinencoded by the transgene to potentially act in the serum. In the case oftreatment for an LSD, the enzyme encoded by the transgene fusion wouldbe able to act on the metabolic products that are accumulating in theserum from its location on the surface of the cell (e.g., RBC). Inaddition, if the RBC is engulfed by a splenic macrophage as is thenormal course of degradation, the lysosome formed when the macrophageengulfs the cell would expose the membrane bound fusion protein to thehigh concentrations of metabolic products in the lysosome at the pH morenaturally favorable to that enzyme. Non-limiting examples of potentialfusion partners are shown below in Table 1.

TABLE 1 Examples of potential fusion partners Name Activity Band 3 Aniontransporter, makes up to 25% of the RBC membrane surface proteinAquaporin 1 water transporter Glut1 glucose and L-dehydroascorbic acidtransporter Kidd antigen protein urea transporter RhAG gas transporterATP1A1, ATP1B1 Na+/K+ - ATPase ATP2B1, ATP2B2, Ca2+ - ATPase ATP2B3,ATP2B4 NKCC1, NKCC2 Na+ K+ 2Cl− - cotransporter SLC12A3 Na+—Cl− -cotransporter SLC12A1, SLA12A2 Na—K - cotransporter KCC1 K—Clcotransporter KCNN4 Gardos Channel

Lysosomal storage diseases typically fall into five classes. Theseclasses are shown below in Table 2 along with specific examples of thediseases. Thus, the donor molecules described herein can includesequences coding for one or more enzymes lacking or deficient insubjects with lysosomal storage diseases, including but not limited tothe proteins shown in Table 2.

TABLE 2 Lysosomal Storage Diseases Disease Protein Disease AssociatedGene Accumulated product 1. DEFECTS IN GLYCAN DEGRADATION I. Defects inglycoprotein degradation α-Sialidase (neuraminidase) Sialidosis NEU1sialidated glycopeptides and oligosaccharides Cathepsin AGalactosialidosis CTSA polysaccharide lysosomal alpha- α-MannosidosisMAN2B1 mannose-rich mannosidase glycoproteins and oligosaccharideslysosomal beta- β-Mannosidosis MANBA mannosidase GlycosylasparaginaseAspartylglucosaminuria AGA glycoasparagines Alpha L FucosidaseFucosidosis FUCA1 fucose α-N-Acetylglucosaminidase Sanfilippo syndrome BNAGLU glycosaminoglycan II. Defects in glycolipid degradation A. GM1Ganglioside β-Galactosidase GM1 gangliosidosis/MPS IVB GLB1 keratansulfate β-Hexosaminidase α- GM2-gangliosidosis (Tay-Sachs) HEXA GM2ganglioside subunit β-Hexosaminidase β- GM2-gangliosidosis (Sandhoff)HEXB GM2 ganglioside subunit GM2 activator protein GM2 gangliosidosisGM2A GM2 ganglioside Glucocerebrosidase Gaucher disease GBAglucocerebroside Saposin C Gaucher disease (atypical) PSAPglucocerebroside B. Defects in the degradation of sulfatideArylsulfatase A Metachromatic leukodystrophy ARSA sulphatide Saposin BMetachromatic leukodystrophy PSAP sulphatide Formyl-Glycin generatingMultiple sulfatase deficiency SUMF1 sulfated lipids enzymeβ-Galactosylceramidase Globoid cell leukodystrophy GALCgalactocerebroside (Krabbe) C. Defects in degradation ofglobotriaosylceramide α-Galactosidase A Fabry GLA globotriaosylceramideIII. Defects in degradation of Glycosaminoglycan (Mucopolysaccharidoses)A. Degradation of heparan sulphate Iduronate sulfatase MPS II (Hunter)IDS Dermatan sulfate, Heparan sulfate Iduronidase MPS 1 (Hurler, Scheie)IDUA Dermatan sulfate, Heparan sulfate Heparan N-sulfatase MPS IIIa(Sanfilippo A) SGSH Heparan sulfate Acetyl-CoA transferase MPS IIIc(Sanfilippo C) HGSNAT Heparan sulfate N-acetyl glucosaminidase MPS IIIb(Sanfilippo B) NAGLU Heparan sulfate β-glucuronidase MPS VII (Sly) GUSBN-acetyl glucosamine 6- MPS IIId (Sanfilippo D) GNS Heparan sulfatesulfatase B. Degradation of other mucopolysaccharides B-GalactosidaseMPS VIB (Morquio B) GLB1 Keratan sulfate, Galactose 6-sulfatase MPS IVA(Morquio A) GALNS Keratan sulfate, Chondroitin 6-sulfate HyaluronidaseMPS IX HYAL1 Hyaluronic acid C. Defects in degradation of glycogenα-Glucosidase Pompe GAA Glycogen 2. DEFECTS IN LIPID DEGRADATION I.Defects in degradation of sphingomyelin Acid sphingomyelinase NiemannPick type A SMPD1 sphingomyelin Acid ceramidase Farberlipogranulomatosis ASAH1 nonsulfonated acid mucopolysaccharide II.Defects in degradation of triglycerides and cholesteryls ester Acidlipase Wolman and cholesteryl ester LIPA cholesteryl esters storagedisease 3. DEFECTS IN PROTEIN DEGRADATION Cathepsin K PycnodystostosisCTSK Tripeptidyl peptidase Ceroide lipofuscinosis PPT2 Palmitoyl-proteinCeroide lipofuscinosis PPT1 thioesterase 4. DEFECTS IN LYSOSOMALTRANSPORTERS Cystinosin (cystin transport) Cystinosis CTNS Sialin(sialic acid transport) Salla disease SLC17A5 N-acetylneuraminic acid 5.DEFECTS IN LYSOSOMAL TRAFFICKING PROTEINS Phosphotransferase γ-Mucolipidosis III (I-cell) GNPTG subunit Mucolipin-1(cationMucolipidosis MCOLN1 channel) LYSOSOME-ASSOCIATED Danon LAMP2 MEMBRANEPROTEIN 2 Niemann-Pick disease, type Niemann Pick type C NPC1 LDLcholesterol C1 palmitoyl-protein Ceroid lipofuscinosis (Batten CLN3autofluorescent thioesterase-1 Disease) lipopigment storage materialneuronal ceroid Ceroid lipofuscinosis 6 CLN 6 lipofuscinosis-6 neuronalceroid Ceroid lipofuscinosis 8 CLN 8 lipofuscinosis-8 LYSOSOMALTRAFFICKING Chediak-Higashi LYST REGULATOR Myocilin Griscelli Type 1MYOC RAS-associated protein 27A Griscelli Type 2 RAB27A MelanophilinGriscelli Type 3 MLPH or MYO5A AP3 β-subunit Hermansky Pudliak AP3B1ceroid

In some cases, the donor may be an endogenous gene that has beenmodified. Although antibody response to enzyme replacement therapyvaries with respect to the specific therapeutic enzyme in question andwith the individual patient, a significant immune response has been seenin many LSD patients being treated with enzyme replacement. In addition,the relevance of these antibodies to the efficacy of treatment is alsovariable (see Katherine Ponder, (2008) J Clin Invest 118(8):2686). Thus,the methods and compositions of the current invention can comprise theuse of donor molecules whose sequence has been altered by functionallysilent amino acid changes at sites known to be priming epitopes forendogenous immune responses, such that the polypeptide produced by sucha donor is less immunogenic.

LSD patients often have neurological sequelae due the lack of themissing enzyme in the brain. Unfortunately, it is often difficult todeliver therapeutics to the brain via the blood due to theimpermeability of the blood brain barrier. Thus, the methods andcompositions of the invention may be used in conjunction with methods toincrease the delivery of the therapeutic into the brain. There are somemethods that cause a transient opening of the tight junctions betweencells of the brain capillaries. Examples include transient osmoticdisruption through the use of an intracarotid administration of ahypertonic mannitol solution, the use of focused ultrasound and theadministration of a bradykinin analogue. Alternatively, therapeutics canbe designed to utilize receptors or transport mechanisms for specifictransport into the brain. Examples of specific receptors that may beused include the transferrin receptor, the insulin receptor or thelow-density lipoprotein receptor related proteins 1 and 2 (LRP-1 andLRP-2). LRP is known to interact with a range of secreted proteins suchas apoE, tPA, PAI-1 etc, and so fusing a recognition sequence from oneof these proteins for LRP may facilitate transport of the enzyme intothe brain, following expression in the liver of the therapeutic proteinand secretion into the blood stream (see Gabathuler, (2010) ibid).

Delivery

The nucleases, polynucleotides encoding these nucleases, donorpolynucleotides and compositions comprising the proteins and/orpolynucleotides described herein may be delivered in vivo or ex vivo byany suitable means.

Methods of delivering nucleases as described herein are described, forexample, in U.S. Pat. Nos. 6,453,242; 6,503,717; 6,534,261; 6,599,692;6,607,882; 6,689,558; 6,824,978; 6,933,113; 6,979,539; 7,013,219; and7,163,824, the disclosures of all of which are incorporated by referenceherein in their entireties.

Nucleases and/or donor constructs as described herein may also bedelivered using vectors containing sequences encoding one or more of thezinc finger or TALEN protein(s). Any vector systems may be usedincluding, but not limited to, plasmid vectors, retroviral vectors,lentiviral vectors, adenovirus vectors, poxvirus vectors; herpesvirusvectors and adeno-associated virus vectors, etc. See, also, U.S. Pat.Nos. 6,534,261; 6,607,882; 6,824,978; 6,933,113; 6,979,539; 7,013,219;and 7,163,824, incorporated by reference herein in their entireties.Furthermore, it will be apparent that any of these vectors may compriseone or more of the sequences needed for treatment. Thus, when one ormore nucleases and a donor construct are introduced into the cell, thenucleases and/or donor polynucleotide may be carried on the same vectoror on different vectors. When multiple vectors are used, each vector maycomprise a sequence encoding one or multiple nucleases and/or donorconstructs.

Conventional viral and non-viral based gene transfer methods can be usedto introduce nucleic acids encoding nucleases and donor constructs incells (e.g., mammalian cells) and target tissues. Non-viral vectordelivery systems include DNA plasmids, naked nucleic acid, and nucleicacid complexed with a delivery vehicle such as a liposome or poloxamer.Viral vector delivery systems include DNA and RNA viruses, which haveeither episomal or integrated genomes after delivery to the cell. For areview of gene therapy procedures, see Anderson, Science 256:808-813(1992); Nabel & Felgner, TIBTECH 11:211-217 (1993); Mitani & Caskey,TIBTECH 11:162-166 (1993); Dillon, TIBTECH 11:167-175 (1993); Miller,Nature 357:455-460 (1992); Van Brunt, Biotechnology 6(10):1149-1154(1988); Vigne, Restorative Neurology and Neuroscience 8:35-36 (1995);Kremer & Perricaudet, British Medical Bulletin 51(1):31-44 (1995);Haddada et al., in Current Topics in Microbiology and ImmunologyDoerfler and Bohm (eds.) (1995); and Yu et al., Gene Therapy 1:13-26(1994).

Methods of non-viral delivery of nucleic acids include electroporation,lipofection, microinjection, biolistics, virosomes, liposomes,immunoliposomes, polycation or lipid:nucleic acid conjugates, naked DNA,artificial virions, and agent-enhanced uptake of DNA. Sonoporationusing, e.g., the Sonitron 2000 system (Rich-Mar) can also be used fordelivery of nucleic acids.

Additional exemplary nucleic acid delivery systems include thoseprovided by Amaxa Biosystems (Cologne, Germany), Maxcyte, Inc.(Rockville, Md.), BTX Molecular Delivery Systems (Holliston, Mass.) andCopernicus Therapeutics Inc, (see for example US6008336). Lipofection isdescribed in e.g., U.S. Pat. Nos. 5,049,386; 4,946,787; and 4,897,355)and lipofection reagents are sold commercially (e.g., Transfectam™ andLipofectin™). Cationic and neutral lipids that are suitable forefficient receptor-recognition lipofection of polynucleotides includethose of Feigner, WO 91/17424, WO 91/16024.

The preparation of lipid:nucleic acid complexes, including targetedliposomes such as immunolipid complexes, is well known to one of skillin the art (see, e.g., Crystal, Science 270:404-410 (1995); Blaese etal., Cancer Gene Ther. 2:291-297 (1995); Behr et al., Bioconjugate Chem.5:382-389 (1994); Remy et al., Bioconjugate Chem. 5:647-654 (1994); Gaoet al., Gene Therapy 2:710-722 (1995); Ahmad et al., Cancer Res.52:4817-4820 (1992); U.S. Pat. Nos. 4,186,183, 4,217,344, 4,235,871,4,261,975, 4,485,054, 4,501,728, 4,774,085, 4,837,028, and 4,946,787).

Additional methods of delivery include the use of packaging the nucleicacids to be delivered into EnGeneIC delivery vehicles (EDVs). These EDVsare specifically delivered to target tissues using bispecific antibodieswhere one arm of the antibody has specificity for the target tissue andthe other has specificity for the EDV. The antibody brings the EDVs tothe target cell surface and then the EDV is brought into the cell byendocytosis. Once in the cell, the contents are released (see MacDiarmidet al (2009) Nature Biotechnology 27(7):643).

The use of RNA or DNA viral based systems for the delivery of nucleicacids encoding engineered ZFPs take advantage of highly evolvedprocesses for targeting a virus to specific cells in the body andtrafficking the viral payload to the nucleus. Viral vectors can beadministered directly to subjects (in vivo) or they can be used to treatcells in vitro and the modified cells are administered to subjects (exvivo). Conventional viral based systems for the delivery of ZFPsinclude, but are not limited to, retroviral, lentivirus, adenoviral,adeno-associated, vaccinia and herpes simplex virus vectors for genetransfer. Integration in the host genome is possible with theretrovirus, lentivirus, and adeno-associated virus gene transfermethods, often resulting in long term expression of the insertedtransgene. Additionally, high transduction efficiencies have beenobserved in many different cell types and target tissues.

The tropism of a retrovirus can be altered by incorporating foreignenvelope proteins, expanding the potential target population of targetcells. Lentiviral vectors are retroviral vectors that are able totransduce or infect non-dividing cells and typically produce high viraltiters. Selection of a retroviral gene transfer system depends on thetarget tissue. Retroviral vectors are comprised of cis-acting longterminal repeats with packaging capacity for up to 6-10 kb of foreignsequence. The minimum cis-acting LTRs are sufficient for replication andpackaging of the vectors, which are then used to integrate thetherapeutic gene into the target cell to provide permanent transgeneexpression. Widely used retroviral vectors include those based uponmurine leukemia virus (MuLV), gibbon ape leukemia virus (GaLV), SimianImmunodeficiency virus (SW), human immunodeficiency virus (HIV), andcombinations thereof (see, e.g., Buchscher et al., J. Virol.66:2731-2739 (1992); Johann et al. J. Virol. 66:1635-1640 (1992);Sommerfelt et al., Virol. 176:58-59 (1990); Wilson et al., J. Virol.63:2374-2378 (1989); Miller et al., J. Virol. 65:2220-2224 (1991);PCT/US94/05700).

In applications in which transient expression is preferred, adenoviralbased systems can be used. Adenoviral based vectors are capable of veryhigh transduction efficiency in many cell types and do not require celldivision. With such vectors, high titer and high levels of expressionhave been obtained. This vector can be produced in large quantities in arelatively simple system. Adeno-associated virus (“AAV”) vectors arealso used to transduce cells with target nucleic acids, e.g., in the invitro production of nucleic acids and peptides, and for in vivo and exvivo gene therapy procedures (see, e.g., West et al., Virology 160:38-47(1987); U.S. Pat. No. 4,797,368; WO 93/24641; Kotin, Human Gene Therapy5:793-801 (1994); Muzyczka, J. Clin. Invest. 94:1351 (1994).Construction of recombinant AAV vectors are described in a number ofpublications, including U.S. Pat. No. 5,173,414; Tratschin et al. Mol.Cell. Biol. 5:3251-3260 (1985); Tratschin, et al., Mol. Cell. Biol.4:2072-2081 (1984); Hermonat & Muzyczka, PNAS 81:6466-6470 (1984); andSamulski et al., J. Virol. 63:03822-3828 (1989).

At least six viral vector approaches are currently available for genetransfer in clinical trials, which utilize approaches that involvecomplementation of defective vectors by genes inserted into helper celllines to generate the transducing agent.

pLASN and MFG-S are examples of retroviral vectors that have been usedin clinical trials (Dunbar et al., Blood 85:3048-305 (1995); Kohn etal., Nat. Med. 1:1017-102 (1995); Malech et al., PNAS 94:22 12133-12138(1997)). PA317/pLASN was the first therapeutic vector used in a genetherapy trial. (Blaese et al., Science 270:475-480 (1995)). Transductionefficiencies of 50% or greater have been observed for MFG-S packagedvectors. (Ellem et al., Immunol Immunother. 44(1):10-20 (1997); Dranoffet al., Hum. Gene Ther. 1:111-2 (1997).

Recombinant adeno-associated virus vectors (rAAV) are a promisingalternative gene delivery systems based on the defective andnonpathogenic parvovirus adeno-associated type 2 virus. All vectors arederived from a plasmid that retains only the AAV 145 bp invertedterminal repeats flanking the transgene expression cassette. Efficientgene transfer and stable transgene delivery due to integration into thegenomes of the transduced cell are key features for this vector system.(Wagner et al., Lancet 351:9117 1702-3 (1998), Kearns et al., Gene Ther.9:748-55 (1996)). Other AAV serotypes, including by non-limitingexample, AAV1, AAV3, AAV4, AAV5, AAV6, AAV8, AAV 8.2, AAV9, and AAV rh10and pseudotyped AAV such as AAV2/8, AAV2/5 and AAV2/6 can also be usedin accordance with the present invention.

Replication-deficient recombinant adenoviral vectors (Ad) can beproduced at high titer and readily infect a number of different celltypes. Most adenovirus vectors are engineered such that a transgenereplaces the Ad E1a, E1b, and/or E3 genes; subsequently the replicationdefective vector is propagated in human 293 cells that supply deletedgene function in trans. Ad vectors can transduce multiple types oftissues in vivo, including non-dividing, differentiated cells such asthose found in liver, kidney and muscle. Conventional Ad vectors have alarge carrying capacity. An example of the use of an Ad vector in aclinical trial involved polynucleotide therapy for anti-tumorimmunization with intramuscular injection (Sterman et al., Hum. GeneTher. 7:1083-9 (1998)). Additional examples of the use of adenovirusvectors for gene transfer in clinical trials include Rosenecker et al.,Infection 24:1 5-10 (1996); Sterman et al., Hum. Gene Ther. 9:71083-1089 (1998); Welsh et al., Hum. Gene Ther. 2:205-18 (1995); Alvarezet al., Hum. Gene Ther. 5:597-613 (1997); Topf et al., Gene Ther.5:507-513 (1998); Sterman et al., Hum. Gene Ther. 7:1083-1089 (1998).

Packaging cells are used to form virus particles that are capable ofinfecting a host cell. Such cells include 293 cells, which packageadenovirus, and ψ2 cells or PA317 cells, which package retrovirus. Viralvectors used in gene therapy are usually generated by a producer cellline that packages a nucleic acid vector into a viral particle. Thevectors typically contain the minimal viral sequences required forpackaging and subsequent integration into a host (if applicable), otherviral sequences being replaced by an expression cassette encoding theprotein to be expressed. The missing viral functions are supplied intrans by the packaging cell line. For example, AAV vectors used in genetherapy typically only possess inverted terminal repeat (ITR) sequencesfrom the AAV genome which are required for packaging and integrationinto the host genome. Viral DNA is packaged in a cell line, whichcontains a helper plasmid encoding the other AAV genes, namely rep andcap, but lacking ITR sequences. The cell line is also infected withadenovirus as a helper. The helper virus promotes replication of the AAVvector and expression of AAV genes from the helper plasmid. The helperplasmid is not packaged in significant amounts due to a lack of ITRsequences. Contamination with adenovirus can be reduced by, e.g., heattreatment to which adenovirus is more sensitive than AAV.

In many gene therapy applications, it is desirable that the gene therapyvector be delivered with a high degree of specificity to a particulartissue type. Accordingly, a viral vector can be modified to havespecificity for a given cell type by expressing a ligand as a fusionprotein with a viral coat protein on the outer surface of the virus. Theligand is chosen to have affinity for a receptor known to be present onthe cell type of interest. For example, Han et al., Proc. Natl. Acad.Sci. USA 92:9747-9751 (1995), reported that Moloney murine leukemiavirus can be modified to express human heregulin fused to gp70, and therecombinant virus infects certain human breast cancer cells expressinghuman epidermal growth factor receptor. This principle can be extendedto other virus-target cell pairs, in which the target cell expresses areceptor and the virus expresses a fusion protein comprising a ligandfor the cell-surface receptor. For example, filamentous phage can beengineered to display antibody fragments (e.g., FAB or Fv) havingspecific binding affinity for virtually any chosen cellular receptor.Although the above description applies primarily to viral vectors, thesame principles can be applied to nonviral vectors. Such vectors can beengineered to contain specific uptake sequences which favor uptake byspecific target cells.

Gene therapy vectors can be delivered in vivo by administration to anindividual patient, typically by systemic administration (e.g.,intravenous, intraperitoneal, intramuscular, subdermal, or intracranialinfusion) or topical application, as described below. Alternatively,vectors can be delivered to cells ex vivo, such as cells explanted froman individual patient (e.g., lymphocytes, bone marrow aspirates, tissuebiopsy) or universal donor hematopoietic stem cells, followed byreimplantation of the cells into a patient, usually after selection forcells which have incorporated the vector.

Vectors (e.g., retroviruses, adenoviruses, liposomes, etc.) containingnucleases and/or donor constructs can also be administered directly toan organism for transduction of cells in vivo. Alternatively, naked DNAcan be administered. Administration is by any of the routes normallyused for introducing a molecule into ultimate contact with blood ortissue cells including, but not limited to, injection, infusion, topicalapplication and electroporation. Suitable methods of administering suchnucleic acids are available and well known to those of skill in the art,and, although more than one route can be used to administer a particularcomposition, a particular route can often provide a more immediate andmore effective reaction than another route.

Vectors suitable for introduction of polynucleotides described hereininclude non-integrating lentivirus vectors (IDLY). See, for example, Oryet al. (1996) Proc. Natl. Acad. Sci. USA 93:11382-11388; Dull et al.(1998) J. Virol. 72:8463-8471; Zuffery et al. (1998) J. Virol.72:9873-9880; Follenzi et al. (2000) Nature Genetics 25:217-222; U.S.Patent Publication No 2009/054985.

Pharmaceutically acceptable carriers are determined in part by theparticular composition being administered, as well as by the particularmethod used to administer the composition. Accordingly, there is a widevariety of suitable formulations of pharmaceutical compositionsavailable, as described below (see, e.g., Remington's PharmaceuticalSciences, 17th ed., 1989).

It will be apparent that the nuclease-encoding sequences and donorconstructs can be delivered using the same or different systems. Forexample, a donor polynucleotide can be carried by a plasmid, while theone or more nucleases can be carried by a AAV vector. Furthermore, thedifferent vectors can be administered by the same or different routes(intramuscular injection, tail vein injection, other intravenousinjection, intraperitoneal administration and/or intramuscularinjection. The vectors can be delivered simultaneously or in anysequential order.

Formulations for both ex vivo and in vivo administrations includesuspensions in liquid or emulsified liquids. The active ingredientsoften are mixed with excipients which are pharmaceutically acceptableand compatible with the active ingredient. Suitable excipients include,for example, water, saline, dextrose, glycerol, ethanol or the like, andcombinations thereof. In addition, the composition may contain minoramounts of auxiliary substances, such as, wetting or emulsifying agents,pH buffering agents, stabilizing agents or other reagents that enhancethe effectiveness of the pharmaceutical composition.

Applications

The methods of this invention contemplate the treatment of a monogenicdisease (e.g. lysosomal storage disease). Treatment can compriseinsertion of the corrected disease associated gene in safe harbor locus(e.g. albumin) for expression of the needed enzyme and release into theblood stream. Insertion into a secretory cell, such as a liver cell forrelease of the product into the blood stream, is particularly useful.The methods and compositions of the invention also can be used in anycircumstance wherein it is desired to supply a transgene encoding one ormore therapeutics in a hemapoietic stem cell such that mature cells(e.g., RBCs) derived from these cells contain the therapeutic. Thesestem cells can be differentiated in vitro or in vivo and may be derivedfrom a universal donor type of cell which can be used for all patients.Additionally, the cells may contain a transmembrane protein to trafficthe cells in the body. Treatment can also comprise use of patient cellscontaining the therapeutic transgene where the cells are developed exvivo and then introduced back into the patient. For example, HSCcontaining a suitable transgene may be inserted into a patient via abone marrow transplant. Alternatively, stem cells such as muscle stemcells or iPSC which have been edited using with the therapeutictransgene maybe also injected into muscle tissue.

Thus, this technology may be of use in a condition where a patient isdeficient in some protein due to problems (e.g., problems in expressionlevel or problems with the protein expressed as sub- ornon-functioning). Particularly useful with this invention is theexpression of transgenes to correct or restore functionality inlysosomal storage disorders.

By way of non-limiting examples, production of the defective or missingproteins accomplished and used to treat the lysosomal storage disease.Nucleic acid donors encoding the proteins may be inserted into a safeharbor locus (e.g. albumin or HPRT) and expressed either using anexogenous promoter or using the promoter present at the safe harbor.Alternatively, donors can be used to correct the defective gene in situ.The desired transgene may be inserted into a CD34+ stem cell andreturned to a patient during a bone marrow transplant. Finally, thenucleic acid donor maybe be inserted into a CD34+ stem cell at a betaglobin locus such that the mature red blood cell derived from this cellhas a high concentration of the biologic encoded by the nucleic aciddonor. The biologic containing RBC can then be targeted to the correcttissue via transmembrane proteins (e.g. receptor or antibody).Additionally, the RBCs may be sensitized ex vivo viaelectrosensitization to make them more susceptible to disruptionfollowing exposure to an energy source (see WO2002007752).

In some applications, an endogenous gene may be knocked out by use ofthe methods and compositions of the invention. Examples of this aspectinclude knocking out an aberrant gene regulator or an aberrant diseaseassociated gene. In some applications, an aberrant endogenous gene maybe replaced, either functionally or in situ, with a wild type version ofthe gene. The inserted gene may also be altered to improve thefunctionality of the expressed protein or to reduce its immunogenicity.In some applications, the inserted gene is a fusion protein to increaseits transport into a selected tissue such as the brain.

The following Examples relate to exemplary embodiments of the presentdisclosure in which the nuclease comprises a zinc finger nuclease (ZFN)or TALEN. It will be appreciated that this is for purposes ofexemplification only and that other nucleases or nuclease systems can beused, for instance homing endonucleases (meganucleases) with engineeredDNA-binding domains and/or fusions of naturally occurring of engineeredhoming endonucleases (meganucleases) DNA-binding domains andheterologous cleavage domains and/or a CRISPR/Cas system comprising anengineered single guide RNA.

EXAMPLES Example 1 Design, Construction and General Characterization ofAlbumin-Specific Nucleases

Zinc finger proteins were designed and incorporated into plasmids, AAVor adenoviral vectors essentially as described in Urnov et al. (2005)Nature 435(7042):646-651, Perez et al (2008) Nature Biotechnology26(7):808-816, and as described in U.S. Pat. No. 6,534,261. Table 3shows the recognition helices within the DNA binding domain of exemplaryalbumin-specific ZFPs while Table 4 shows the target sites for theseZFPs (see co-owned U.S. Provisionals 61/537,349 and 61/560,506).Nucleotides in the target site that are contacted by the ZFP recognitionhelices are indicated in uppercase letters; non-contacted nucleotidesindicated in lowercase. Albumin-specific TALENs were also designed andare set forth in U.S. application Ser. Nos. 13/624,193 and 13/624,217and incorporated by reference in their entireties).

TABLE 3 Murine albumin-specific zinc finger nucleases helix designsDesign Target SBS # F1 F2 F3 F4 F5 F6 Intron 1 30724 TSGSLTR RSDALSTQSATRTK TSGHLSR QSGNLAR NA (SEQ ID (SEQ ID (SEQ ID (SEQ ID (SEQ IDNO: 1) NO: 2) NO: 3) NO: 4) NO: 5) Intron 1 30725 RSDHLSA TKSNRTKDRSNLSR WRSSLRA DSSDRKK NA (SEQ ID (SEQ ID (SEQ ID (SEQ ID (SEQ IDNO: 6) NO: 7) NO: 8) NO: 9) NO: 10) Intron 1 30732 TSGNLTR DRSTRRQTSGSLTR ERGTLAR TSANLSR NA (SEQ ID (SEQ ID (SEQ ID (SEQ ID (SEQ IDNO: 11) NO: 12) NO: 1) NO: 13) NO: 14) Intron 1 30733 DRSALAR RSDHLSEHRSDRTR QSGALAR QSGHLSR NS (SEQ ID (SEQ ID (SEQ ID (SEQ ID (SEQ IDNO: 15) NO: 16) NO: 17) NO: 18) NO: 19) Intron 13 30759 RSDNLST DRSALARDRSNLSR DGRNLRH RSDNLAR QSNALNR (SEQ ID (SEQ ID (SEQ ID (SEQ ID (SEQ ID(SEQ ID NO: 20) NO: 15) NO: 8) NO: 21) NO: 22) NO: 23) Intron 13 30761DRSNLSR LKQVLVR QSGNLAR QSTPLFA QSGALAR NA (SEQ ID (SEQ ID (SEQ ID(SEQ ID (SEQ ID NO: 8) NO: 24) NO: 5) NO: 25) NO: 18) Intron 13 30760DRSNLSR DGRNLRH RSDNLAR QSNALNR NA NA (SEQ ID (SEQ ID (SEQ ID (SEQ IDNO: 8) NO: 21) NO: 22) NO: 23) Intron 13 30767 RSDNLSV HSNARKT RSDSLSAQSGNLAR RSDSLSV QSGHLSR (SEQ ID (SEQ ID (SEQ ID (SEQ ID (SEQ ID (SEQ IDNO: 26) NO: 27) NO: 28) NO: 5) NO: 29) NO: 19) Intron 13 30768 RSDNLSEERANRNS QSANRTK ERGTLAR RSDALTQ NA (SEQ ID (SEQ ID (SEQ ID (SEQ ID(SEQ ID NO: 30) NO: 31) NO: 32) NO: 13) NO: 33) Intron 13 30769 TSGSLTRDRSNLSR DGRNLRH ERGTLAR RSDALTQ NA (SEQ ID (SEQ ID (SEQ ID (SEQ ID(SEQ ID NO: 1) NO: 8) NO: 21) NO: 13) NO: 33) Intron 12 30872 QSGHLARRSDHLTQ RSDHLSQ WRSSLVA RSDVLSE RNQHRKT (SEQ ID (SEQ ID (SEQ ID (SEQ ID(SEQ ID (SEQ ID NO: 34) NO: 35) NO: 36) NO: 37) NO: 38) NO: 39)Intron 12 30873 QSGDLTR RSDALAR QSGDLTR RRDPLIN RSDNLSV IRSTLRD (SEQ ID(SEQ ID (SEQ ID (SEQ ID (SEQ ID (SEQ ID NO: 40) NO: 41) NO: 40) NO: 42)NO: 26) NO: 43) Intron 12 30876 RSDNLSV YSSTRNS RSDHLSA SYWSRTV QSSDLSRRTDALRG (SEQ ID (SEQ ID (SEQ ID (SEQ ID (SEQ ID (SEQ ID NO: 26) NO: 44)NO: 6) NO: 45) NO: 46) NO: 47) Intron 12 30877 RSDNLST QKSPLNT TSGNLTRQAENLKS QSSDLSR RTDALRG (SEQ ID (SEQ ID (SEQ ID (SEQ ID (SEQ ID (SEQ IDNO: 20) NO: 48) NO: 11) NO: 49) NO: 46) NO: 47) Intron 12 30882 RSDNLSVRRAHLNQ TSGNLTR SDTNRFK RSDNLST QSGHLSR (SEQ ID (SEQ ID (SEQ ID (SEQ ID(SEQ ID (SEQ ID NO: 26) NO: 50) NO: 11) NO: 51) NO: 20) NO: 19)Intron 12 30883 DSSDRKK DRSALSR TSSNRKT QSGALAR RSDHLSR NA (SEQ ID(SEQ ID (SEQ ID (SEQ ID (SEQ ID NO: 10) NO: 52) NO: 53) NO: 18) NO: 54)

TABLE 4 Target sites of murine albumin-specific ZFNs Target SBS #Target site Intron 1 30724 ctGAAGGTgGCAATGGTTcctctctgct_ (SEQ ID NO: 55)Intron 1 30725 ttTCCTGTAACGATCGGgaactggcatc_ (SEQ ID NO: 56) Intron 130732 aaGATGCCaGTTCCCGATcgttacagga_ (SEQ ID NO: 57) Intron 1 30733agGGAGTAGCTTAGGTCagtgaagagaa_ (SEQ ID NO: 58) Intron 13 30759acGTAGAGAACAACATCTAGattggtgg_ (SEQ ID NO: 59) Intron 13 30761ctGTAATAGAAACTGACttacgtagctt_ (SEQ ID NO: 60) Intron 13 30760acGTAGAGAACAACatctagattggtgg_ (SEQ ID NO: 59) Intron 13 30767agGGAATGtGAAATGATTCAGatatata_ (SEQ ID NO: 61) Intron 13 30768ccATGGCCTAACAACAGtttatcttctt_ (SEQ ID NO: 62) Intron 13 30769ccATGGCCtAACAACaGTTtatcttctt_ (SEQ ID NO: 62) Intron 12 30872ctTGGCTGTGTAGGAGGGGAgtagcagt_ (SEQ ID NO: 63) Intron 12 30873ttCCTAAGTTGGCAGTGGCAtgcttaat_ (SEQ ID NO: 64) Intron 12 30876ctTTGGCTTTGAGGATTAAGcatgccac_ (SEQ ID NO: 65) Intron 12 30877acTTGGCTcCAAGATTTATAGccttaaa_ (SEQ ID NO: 66) Intron 12 30882caGGAAAGTAAGATAGGAAGgaatgtga_ (SEQ ID NO: 67) Intron 12 30883ctGGGGTAAATGTCTCCttgctcttctt_ (SEQ ID NO: 68)

Example 2 Activity of Murine Albumin-Specific Nucleases

ZFN pairs targeting the murine albumin gene were used to test theability of these ZFNs to induce DSBs at a specific target site. Theamino acid sequence of the recognition helix region of the indicatedZFNs are shown below in Table 3 and their target sites shown in Table 4(DNA target sites indicated in uppercase letters; non-contactednucleotides indicated in lowercase).

The Cel-I assay (Surveyor™, Transgenomics) as described in Perez et al,(2008) Nat. Biotechnol. 26: 808-816 and Guschin et al, (2010) MethodsMol Biol. 649:247-56), was used to detect ZFN-induced modifications. Inthis assay, PCR-amplification of the target site was followed byquantification of insertions and deletions (indels) using the mismatchdetecting enzyme Cel-I (Yang et al, (2000) Biochemistry 39, 3533-3541)which provided a lower-limit estimate of DSB frequency. Three daysfollowing transfection of the ZFN expression vector at either standardconditions (37° C.) or using a hypothermic shock (30° C., see co-ownedU.S. Publication No. 20110041195), genomic DNA was isolated from Neuro2Acells transfected with the ZFN(s) using the DNeasy™ kit (Qiagen). Inthese experiments, all ZFN pairs were ELD/KKR FokI mutation pairs(described above).

A composite of the results from the Cel-I assay are shown in FIG. 1, anddemonstrate that the ZFNs shown below are capable of inducing cleavageat their respective target sites. The percent indels shown beneath thelanes indicates the amount of genomes that were altered by NHEJfollowing cleavage. The data also demonstrates increased activity whenthe transduction procedure incorporates the hypothermic shock.

Example 3 In Vivo Cleavage of the Albumin Locus

The mouse albumin specific ZFNs SBS30724 and SBS30725 which target asequence in intron 1 were tested in vivo. The ZFNs were introduced intoan AAV2/8 vector as described previously (Li et al (2011) Nature 475(7355): 217). To facilitate production in the baculovirus system, thevector AAV2/8.2 was used for preparations destined for baculoviralproduction. AAV2/8.2 differs from the AAV2/8 vector in that a portion ofthe AAV8 capsid has been removed and replaced by a same region from theAAV2 capsid creating a chimeric capsid. The region is the phospholipaseA2 domain in VP 1. Production of the ZFN containing virus particles wasdone either by preparation using a HEK293 system or a baculovirus systemusing standard methods in the art (See Li et al, ibid, see e.g.US6723551). The virus particles were then administered to normal malemice (n=6) using a single dose of 200 microliter of 1.0e11 total vectorgenomes of either AAV2/8 or AAV2/8.2 encoding the mouse albumin-specificZFN. 14 days post administration of rAAV vectors, mice were sacrificed,livers harvested and processed for DNA or total proteins using standardmethods known in the art. Detection of AAV vector genome copies wasperformed by quantitative PCR. Briefly, qPCR primers were made specificto the bGHpA sequences within the AAV as follows:

(SEQ ID NO: 69) Oligo200 (Forward) 5′-GTTGCCAGCCATCTGTTGTTT-3′(SEQ ID NO: 70) Oligo201 (Reverse) 5′-GACAGTGGGAGTGGCACCTT-3′(SEQ ID NO: 71) Oligo202 (Probe) 5′-CTCCCCCGTGCCTTCCTTGACC-3′

Cleavage activity of the ZFN was measured using a Cel-1 assay performedusing a LC-GX apparatus (Perkin Elmer), according to manufacturer'sprotocol. Expression of the ZFNs in vivo was measured using a FLAG-Tagsystem according to standard methods. The results (FIG. 3) demonstratedthat the ZFNs were expressed, and that they are active in cleaving thetarget in the mouse liver gene. Shown in the Figure are the Cel-I NHEJresults for each mouse in the study. The type of vector and theircontents are shown above the lanes. Mismatch repair following ZFNcleavage (indicated % indels) was detected at nearly 16% in some of themice.

Albumin-specific TALENs were also tested as set forth in U.S.application Ser. No. 13/624,193 and 13/624,217 and incorporated byreference in their entireties).

Example 4 In Vivo Insertion of a Corrected Disease Associated Gene

The murine specific albumin ZFNs or TALENs are then used to introducetransgene encoding a therapeutic gene product into the albumin locus forexpression. Donors were designed to insert the correct gene for Fabry'sdisease (GLA), Gaucher's disease (GBA), Hurler's disease (IDUA), andHunter's disease into the albumin locus. In these donor constructs, thetherapeutic gene was flanked by sequences homologous to the albumingene. 5′ of the transgene, the donor constructs all contain sequenceshomologous to the murine albumin intron 1, while 3′ of the gene, theconstructs contain sequences homologous to the murine albumin intron1-exon 2 boundary.

The donor constructs are then incorporated into an AAV genome and theresulting AAV particles containing the donors are then purified usingmethods know in the art. The material is used to produce AAV virusescontaining AAV-donor genomes using the triple transfection method intoHEK 293T cells and purified on CsCl density gradients as has beendescribed (see Ayuso et al. (2010) Gene Ther 17(4), 503-510). AAV vectorwill be diluted in PBS prior to injection. A range of 5e9 to 5e13 v.g.AAV-donor vector particles will be used in conjunction with 1e9 to 1e12vg of AAV-ZFN vector particles via tail vein or intraperitonealinjections of the viruses in wild-type, or disease model mice. AAV-ZFNgenomes, described previously, containing the mouse albumin-specificZFNs will be used, in combination with the GLA, GBA, IDUA and IDSAAV-donors. Cel-I and PCR assays will be performed on liver DNA isolatedat various time points to determine the frequency of NHEJ and ZFNinduced donor insertion. Southern blots may also be used. As perstandard protocol, quantification of human GLA, GBA, IDUA and IDS inplasma will be performed using a human GLA, GBA, IDUA and IDS ELISA kitor using a FLAG Tag ELISA kit. Standard Western blots are alsoperformed. The results demonstrate that these corrective transgenes canincrease the expression of the therapeutic protein in vivo.

For example, the gene encoding human alpha galatosidase (deficient inpatients with Fabry's disease) was inserted into the mouse albuminlocus. The ZFN pair 30724/30725 was used as described above using aalpha galactosidase transgene. In this experiment, 3 mice were treatedwith an AAV2/8 virus containing the ZFN pair at a dose of 3.0e11 viralgenomes per mouse and an AAV2/8 virus containing the huGLa donor at1.5e12 viral genomes per mouse. Control animals were given either theZFN containing virus alone or the huGLa donor virus alone. Western blotsdone on liver homogenates showed an increase in alphagalactosidase-specific signal, indicating that the alpha galactosidasegene had been integrated and was being expressed (FIG. 4A). In addition,an ELISA was performed on the liver lysate using a human alphagalactosidase assay kit (Sino) according to manufacturer's protocol. Theresults, shown in FIG. 4B, demonstrated an increase in signal in themice that had been treated with both the ZFNs and the huGLa donor.

Example 5 Design of Human Albumin Specific ZFNs

To design ZFNs with specificity for the human albumin gene, the DNAsequence of human albumin intron 1 was analyzed using previouslydescribed methods to identify target sequences with the best potentialfor ZFN binding (for details, see co-owned United Stated patentapplication Ser. No. 13/624,193 and 13/624,217). The target and helicesare shown in Tables 5 and 6.

TABLE 5 Human albumin-specific zinc finger nucleases helix designsDesign Target SBS # F1 F2 F3 F4 F5 F6 Intron 1 35393 QSSDLSR LRHNLRADQSNLRA RPYTLRL QSSDLSR HRSNLNK (SEQ ID (SEQ ID (SEQ ID (SEQ ID (SEQ ID(SEQ ID NO: 46) NO: 72) NO: 73) NO: 74) NO: 46) NO: 75) Intron 1 35394QSSDLSR HRSNLNK DQSNLRA RPYTLRL QSSDLSR HRSNLNK (SEQ ID (SEQ ID (SEQ ID(SEQ ID (SEQ ID (SEQ ID NO: 46) NO: 75) NO: 73) NO: 74) NO: 46) NO: 75)Intron 1 35396 QSSDLSR LKWNLRT DQSNLRA RPYTLRL QSSDLSR HRSNLNK (SEQ ID(SEQ ID (SEQ ID (SEQ ID (SEQ ID (SEQ ID NO: 46) NO: 76) NO: 73) NO: 74)NO: 46) NO: 75) Intron 1 35398 QSSDLSR LRHNLRA DQSNLRA RPYTLRL QSSDLSRHRSNLNK (SEQ ID (SEQ ID (SEQ ID (SEQ ID (SEQ ID (SEQ ID NO: 46) NO: 72)NO: 73) NO: 74) NO: 46) NO: 75) Intron 1 35399 QSSDLSR HRSNLNK DQSNLRARPYTLRL QSSDLSR HRSNLNK (SEQ ID (SEQ ID (SEQ ID (SEQ ID (SEQ ID (SEQ IDNO: 46) NO: 75) NO: 73) NO: 74) NO: 46) NO: 75) Intron 1 35405 QSSDLSRWKWNLRA DQSNLRA RPYTLRL QSSDLSR HRSNLNK (SEQ ID (SEQ ID (SEQ ID (SEQ ID(SEQ ID (SEQ ID NO: 46) NO: 77) NO: 73) NO: 74) NO: 46) NO: 75) Intron 135361 QSGNLAR LMQNRNQ LKQHLNE TSGNLTR RRYYLRL N/A (SEQ ID (SEQ ID(SEQ ID (SEQ ID (SEQ ID NO: 5) NO: 78) NO: 79) NO: 11) NO: 80) Intron 135364 QSGNLAR HLGNLKT LKQHLNE TSGNLTR RRDWRRD N/A (SEQ ID (SEQ ID(SEQ ID (SEQ ID (SEQ ID NO: 5) NO: 81) NO: 79) NO: 11) NO: 82) Intron 135370 QSGNLAR LMQNRNQ LKQHLNE TSGNLTR RRDWRRD N/A (SEQ ID (SEQ ID(SEQ ID (SEQ ID (SEQ ID NO: 5) NO: 78) NO: 79) NO: 11) NO: 82) Intron 135379 QRSNLVR TSSNRKT LKHHLTD TSGNLTR RRDWRRD N/A (SEQ ID (SEQ ID(SEQ ID (SEQ ID (SEQ ID NO: (83) NO: 53) NO: 84) NO: 11) NO: 82)Intron 1 35458 DKSYLRP TSGNLTR HRSARKR QSSDLSR WRSSLKT N/A (SEQ ID(SEQ ID (SEQ ID (SEQ ID (SEQ ID NO: 85) NO: 11) NO: 86) NO: 46) NO: 87)Intron 1 35480 TSGNLTR HRSARKR QSGDLTR NRHHLKS N/A N/A (SEQ ID (SEQ ID(SEQ ID (SEQ ID NO: 11) NO: 86) NO: 40) NO: 88) Intron 1 35426 QSGDLTRQSGNLHV QSAHRKN STAALSY TSGSLSR RSDALAR (SEQ ID (SEQ ID (SEQ ID (SEQ ID(SEQ ID (SEQ ID NO: 40) NO: 89) NO: 90) NO: 91) NO: 92) NO: 41) Intron 135428 QSGDLTR QRSNLNI QSAHRKN STAALSY DRSALSR RSDALAR (SEQ ID (SEQ ID(SEQ ID (SEQ ID (SEQ ID (SEQ ID NO: 40) NO: 93) NO: 90) NO: 91) NO: 52)NO: 41) Intron 1 34931 QRTHLTQ DRSNLTR QSGNLAR QKVNRAG N/A N/A (SEQ ID(SEQ ID (SEQ ID (SEQ ID NO: 94) NO: 95) NO: 5) NO: 96) Intron 1 33940RSDNLSV QNANRIT DQSNLRA QSAHRIT TSGNLTR HRSARKR (SEQ ID (SEQ ID (SEQ ID(SEQ ID (SEQ ID (SEQ ID NO: 26) NO: 97) NO: 73) NO: 98) NO: 11) NO: 86)

TABLE 6 Target sites of Human albumin-specific ZFNs Target SBS #Target site Intron 1 35393 ccTATCCATTGCACTATGCTttatttaa (SEQ ID NO: 99)(locus 2) Intron 1 35394 ccTATCCATTGCACTATGCTttatttaa (SEQ ID NO: 99)(locus 2) Intron 1 35396 ccTATCCATTGCACTATGCTttatttaa (SEQ ID NO: 99)(locus 2) Intron 1 35398 ccTATCCATTGCACTATGCTttatttaa (SEQ ID NO: 99)(locus 2) Intron 1 35399 ccTATCCATTGCACTATGCTttatttaa (SEQ ID NO: 99)(locus 2) Intron 1 35405 ccTATCCATTGCACTATGCTttatttaa (SEQ ID NO: 99)(locus 2) Intron 1 35361 ttTGGGATAGTTATGAAttcaatcttca (SEQ ID NO: 100)(locus 2) Intron 1 35364 ttTGGGATAGTTATGAAttcaatcttca (SEQ ID NO: 100)(locus 2) Intron 1 35370 ttTGGGATAGTTATGAAttcaatcttca (SEQ ID NO: 100)(locus 2) Intron 1 35379 ttTGGGATAGTTATGAAttcaatcttca (SEQ ID NO: 100)(locus 2) Intron 1 35458 ccTGTGCTGTTGATCTCataaatagaac (SEQ ID NO: 101)(locus 3) Intron 1 35480 ccTGTGCTGTTGATctcataaatagaac (SEQ ID NO: 101)(locus 3) Intron 1 35426 ttGTGGTTTTTAAAtAAAGCAtagtgca (SEQ ID NO: 102)(locus 3) Intron 1 35428 ttGTGGTTTTTAAAtAAAGCAtagtgca (SEQ ID NO:102)(locus 3) Intron 1 34931 acCAAGAAGACAGActaaaatgaaaata (SEQ ID NO: 103)(locus 4) Intron 1 33940 ctGTTGATAGACACTAAAAGagtattag (SEQ ID NO: 104)(locus 4)

These nucleases were tested in pairs to determine the pair with thehighest activity. The resultant matrices of tested pairs are shown inTables 7 and 8, below where the ZFN used for the right side of the dimeris shown across the top of each matrix, and the ZFN used for the leftside of the dimer is listed on the left side of each matrix. Theresultant activity, as determined by percent of mismatch detected usingthe Cel-I assay is shown in the body of both matrices:

TABLE 7 Activity of Human albumin-specific ZFNs (% mutated targets)35393 35394 35396 35398 35399 35405 ave. 35361 18 19 25 22 23 21 2135364 n.d. 24 23 19 21 21 22 35370 21 19 22 n.d. 22 23 21 35379 21 21n.d. 19 19 21 20

TABLE 8 Activity of Human albumin-specific ZFNs (% mutated targets))35458 35480 ave. 35426 4.5 7 3 35428 4.9 6 3.6 (note: ‘n.d.’ means theassay on this pair was not done)

Thus, highly active nucleases have been developed that recognize targetsequences in intron 1 of human albumin.

Example 6 Design of Albumin Specific TALENs

TALENs were designed to target sequences within human albumin intron 1.Base recognition was achieved using the canonical RVD-basecorrespondences (the “TALE code: NI for A, HD for C, NN for G (NK inhalf repeat), NG for T). TALENs were constructed as previously described(see co-owned U.S. Patent Publication No. 20110301073). Targets for asubset of TALENs were conserved in cynomolgus monkey and rhesus macaquealbumin genes (for details, see co-owned U.S. patent application Ser.Nos. 13/624,193 and 13/624,217). The TALENs were constructed in the“+17” and “+63” TALEN backbones as described in US20110301073. Thetargets and numeric identifiers for the TALENs tested are shown below inTable 9.

TABLE 9 Albumin specific TALENs SBS # site # of RVDs SEQ ID NO: 102249gtTGAAGATTGAATTCAta 15 105 102250 gtTGAAGATTGAATTCATAac 17 106 102251gtGCAATGGATAGGTCTtt 15 107 102252 atAGTGCAATGGATAGGtc 15 108 102253atTGAATTCATAACTATcc 15 109 102254 atTGAATTCATAACTATCCca 17 110 102255atAAAGCATAGTGCAATGGat 17 111 102256 atAAAGCATAGTGCAATgg 15 112 102257ctATGCTTTATTTAAAAac 15 113 102258 ctATGCTTTATTTAAAAACca 17 114 102259atTTATGAGATCAACAGCAca 17 115 102260 ctATTTATGAGATCAACAGca 17 116 102261ttCATTTTAGTCTGTCTTCtt 17 117 102262 atTTTAGTCTGTCTTCTtg 15 118 102263ctAATACTCTTTTAGTGTct 16 119 102264 atCTAATACTCTTTTAGTGtc 17 120 102265atAATTGAACATCATCCtg 15 121 102266 atAATTGAACATCATCCTGag 17 122 102267atATTGGGCTCTGATTCCTac 17 123 102268 atATTGGGCTCTGATTCct 15 124 102269ttTTTCTGTAGGAATCAga 15 125 102270 ttTTTCTGTAGGAATCAGag 16 126 102271ttATGCATTTGTTTCAAaa 15 127 102272 atTATGCATTTGTTTCAaa 15 128

The TALENs were then tested in pairs in HepG2 cells for the ability toinduce modifications at their endogenous chromosomal targets, and theresults showed that many proteins bearing the +17 truncation point wereactive. Similarly, many TALENs bearing the +63 truncation point werealso active (see Table 10). Side by side comparisons with three sets ofnon-optimized albumin ZFNs (see Table 10) showed that the TALENs andZFNs have activities that are in the same approximate range.

TABLE 10 TALEN-induced target modification in HepG2-C3a cells Sample %modification, % pair TALEN C17 C17 TALEN C63 modification, C63 Gap 1102251:102249 15 102251:102249 0 12 2 102251:102250 0 102251:102250 0 103 102252:102249 0 102252:102249 8.3 15 4 102252:102250 32 102252:1022508.0 13 5 102255:102253 38 102255:102253 21 13 6 102255:102254 43102255:102254 0 11 7 102256:102253 0 102256:102253 23 15 8 102256:10225428 102256:102254 16 13 9 102259:102257 18 102259:102257 15 13 10102259:102258 15 102259:102258 0 11 11 102260:102257 15 102260:102257 1315 12 102260:102258 24 102260:102258 11 13 13 102263:102261 0102263:102261 16 17 14 102263:102262 0 102263:102262 15 16 15102264:102261 0 102264:102261 22 18 16 102264:102262 0 102264:102262 1717 20 102267:102265 47 102267:102265 9.8 13 21 102267:102266 4.7102267:102266 0 11 22 102268:102265 4.2 102268:102265 7.9 15 23102268:102266 10 102268:102266 0 13 24 102271:102269 14 102271:102269 012 25 102271:102270 0 102271:102270 0 11 26 102272:102269 0102272:102269 0 13 27 102272:102270 0 102272:102270 0 12 ZFNs 1735361:35396 31 35361:35396 29 6 18 35426:35458 10 35426:35458 7 6 1934931:33940 7.3 34931:33940 7 6

As noted previously (see co-owned U.S. Patent Publication No.20110301073), the C17 TALENs have greater activity when the gap sizebetween the two TALEN target sites is approximately 11-15 bp, while theC63 TALENs sustain activity at gap sizes up to 18 bp (see Table 10).

Example 7 Detection of LSD Donor Transgenes In Vivo

Donors for four lysosomal storage disease transgenes were constructedfor the purpose of integrating into the mouse albumin gene in intron1.The transgenes were α-galactosidase A (GLA), Acid 3-glucosidase (GBA),α-L-iduronidase (IDUA) and Iduronate-2-sulfatase (IDS), genes lacking inFabry's, Gaucher's, Hurler's and Hunter's diseases, respectively. See,e.g., FIG. 8.

The donors were then used in in vivo studies to observe integration ofthe transgenes. The murine albumin specific ZFNs and the donors wereinserted all into AAV2/8 virus as described in Example 4, and then wereinjected into mice. In these experiments, the virus was formulated forinjection in D-PBS+35 mM NaCl, 5% glycerol and frozen prior toinjection. The donor- and nuclease-containing viruses were mixedtogether prior to freezing. At Day 0, the virus preparation was thawedand administered to the mice by tail vein injection where the totalinjection volume was 200 pt. At the indicated times, the mice weresacrificed and then serum, liver and spleen were harvested for proteinand DNA analysis by standard protocols. The dose groups are shown belowin Table 11.

TABLE 11 Treatment groups for LSD transgene integration N/group/ GroupTreatment time point 1 murine Alb intron 1 + Fabry@ 1:5 ratio; ZFN @ 33.0e11, Donor @ 1.5e12 2 murine Alb intron 1 + Hunters donor@ 1:5 ratio;3 ZFN @3.0e11, Donor @ 1.5e12 3 murine Alb intron 1 + Hurlers donor@ 1:5ratio; 3 LEN @3.0e11, Donor @ 1.5e12 4 murine Alb intron 1 3 5 Fabrydonor only 2 6 Hunter's donor only 2 7 Hurler's donor only 2

At day 30, liver homogenates were analyzed by Western blot analysis forthe presence of the LSD proteins encoded by the donors. Liverhomogenates were analyzed by Western blot using standard methodologies,using the following primary antibodies: α-Galactosidase A(Fabry's)-specific rabbit monoclonal antibody was purchased from SinoBiological, Inc.; Human α-L-Iduronidase (Hurler's)—specific mousemonoclonal antibody was purchased from R&D Systems; Human iduronate2-Sulfatase (Hunter's)-specific mouse monoclonal was purchased from R&DSystems. The results (See FIG. 5) demonstrate expression, especially inthe mice that received both the ZFN containing virus and the transgenedonor containing virus.

The manner of integration of the donor DNA into the albumin locus wasalso investigated. Following the cleavage at the albumin locus, thedonor transgene could be potentially be integrated via homology directedrecombination (HDR), utilizing the regions of homology flanking thetransgene donor (FIG. 2), or the transgene may be captured during theerror-prone non-homologous end joining process (NHEJ). The results ofthese two alternatives will yield insertions of differing sizes whensubject to PCR, using either the Acc651-SA-rev-sh primer (5′AAG AAT AATTCT TTA GTG GTA 3′, SEQ ID NO:129) which binds to the F9 splice acceptorsite in all LSD donor constructs and the mALB-OOF1 primer (5′ATGAAGTGGGTAACCTTTCTC 3′, SEQ ID NO:130) which binds to the mousealbumin exon 1 upstream of the ZFN cleavage site (see FIG. 6), where theintegration of the transgene by HDR will result in insertion ofapproximately 680 bp while integration via NHEJ will result inintegration of approximately 1488 bp. Thus, genomic DNA isolated fromthe treated mice liver homogenates was subject to PCR in the presence of³²P-radiolabeled nucleotides and run on a gel. In all three of thetransgene integrations, integration via both mechanisms was observed(see FIG. 7).

Donor DNAs were also designed to include a tag sequence for laterrecognition of the protein using the tag specific antibodies. The tagwas designed to incorporate two different sequences encoding the Myc(EQKLISEEDL, SEQ ID NO:131) and the Flag (DYKDDDD SEQ ID NO:132) tags.Schematics of the donor designs are shown in FIG. 8. The donors wereintegrated as described above, and all were capable of integration asdemonstrated by PCR (see FIG. 9).

All patents, patent applications and publications mentioned herein arehereby incorporated by reference in their entirety.

Although disclosure has been provided in some detail by way ofillustration and example for the purposes of clarity of understanding,it will be apparent to those skilled in the art that various changes andmodifications can be practiced without departing from the spirit orscope of the disclosure. Accordingly, the foregoing descriptions andexamples should not be construed as limiting.

What is claimed is:
 1. A method for generating a cell that produces a protein that treats a lysosomal storage disease, the method comprising: integrating a transgene encoding the protein into an endogenous locus of the cell using a non-naturally occurring nuclease, such that the cell produces the protein that treats a lysosomal storage disease.
 2. The method of claim 1, wherein the lysosomal storage disease is selected from the group consisting of Gaucher's, Fabry's, Hunter's, Hurler's, and Niemann-Pick's.
 3. The method of claim 1, wherein the transgene encodes a protein selected from the group consisting of glucocerebrosidase, α galactosidase, iduronate-2-sulfatase deficiency, alpha-L iduronidase deficiency and sphingomyelin phosphodiesterase.
 4. The method of claim 1, wherein the endogenous locus is selected from the group consisting of an albumin gene, an AAVS1 gene, an HRPT gene, a CCR5 gene, a Rosa gene and a globin gene.
 5. The method of claim 1, wherein expression of the transgene is driven by an endogenous promoter.
 6. The method of claim 1, wherein the transgene is a fusion protein comprising amino acids encoded by the transgene and by the endogenous locus into which the transgene is integrated.
 7. The method of claim 1, wherein the transgene encodes a fusion protein comprising an extracellular domain of a membrane protein such that upon expression, the fusion protein is localized on the surface of the cell.
 8. The method of claim 1, wherein the transgene encodes a fusion protein comprising a ligand for a receptor such that upon expression, the fusion protein crosses the blood brain barrier.
 9. The method of claim 1, wherein the cell is selected from the group consisting of a red blood cell, a liver cell, a muscle cell and a stem cell.
 10. The method of claim 9, wherein the stem cell is a hematopoietic stem cell or induced pluripotent stem cell.
 11. A method of treating a lysosomal storage disease, the method comprising isolating the protein produced by the cell generated according to the method of claim 1, and administering the protein to a patient in need thereof.
 12. A method of treating a lysosomal storage disease, the method comprising administering the cell generated by the method of claim 1 to a subject in need thereof.
 13. A method of treating a lysosomal storage disease, the method comprising administering one or more nucleases and one or more transgenes to a subject in need thereof, such that a cell generated according to the method of claim 1 is generated in the subject.
 14. The method of claim 12, wherein the cell is an isolated stem or precursor cell, and the method further comprises expanding and differentiating the stem or precursor cell prior to administration.
 15. The method of claim 1, wherein the transgene is delivered to the cell using a viral vector.
 16. The method of claim 15, wherein the viral vector is an AAV vector. 